Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl and the Metabolite Norfentanyl in Rat Urine by LC-MS-MS.

Fentanyl and its analogs are potent synthetic opioids with a high potential for abuse and dependence. They have become major contributors to opioid deaths. This study aimed to determine whether the metabolites of fentanyl, alpha-methylfentanyl and beta-hydroxyfentanyl, excreted in the urine, can demonstrate historical drug exposure. Fentanyl is primarily metabolized via CYP3A4 into norfentanyl, although there is little research on its metabolization into alpha-methylfentanyl and beta-hydroxyfentanyl. We conducted in vitro experiments with human liver microsomes (HLMs) and rat liver microsomes (RLMs) to elucidate the major metabolic pathways of alpha-methylfentanyl and beta-hydroxyfentanyl using ultra-high-performance liquid chromatography coupled with mass spectrometry. The results showed that both alpha-methylfentanyl and beta-hydroxyfentanyl were predominantly metabolized into norfentanyl in HLM and RLM. Urine samples were collected at different intervals from 0 h to 72 h after intravenous administration of alpha-methylfentanyl and beta-hydroxyfentanyl (20 µg/kg) to Sprague-Dawley rats. We prepared the samples by liquid-liquid extraction, and the internal standard (IS) was cariprazine. A sensitive, rapid liquid chromatography-tandem mass spectrometry method was developed and validated to determine four analytes in the urine. The lower limit of qualification in urine was 2 pg/mL for fentanyl, 5 pg/mL for alpha-methylfentanyl, 10 pg/mL for beta-hydroxyfentanyl and 40 pg/mL for norfentanyl. The analytical range was 0.002-2 ng/mL for fentanyl, 0.005-5 ng/mL for alpha-methylfentanyl, 0.01-10 ng/mL for beta-hydroxyfentanyl and 0.04-40 ng/mL for norfentanyl. All analytes demonstrated good linearity (R2 > 0.99). The extraction recoveries were in the 67.8%-92.1% range, and the IS-normalized matrix effects were between 55.5% and 74.0% (coefficient of variance < 15%). Our data indicated that norfentanyl has a higher concentration in rat urine and was detectable for at least 3 days after exposure to these compounds. This developed method may be useful in various fields, including forensic analysis, workplace drug testing and monitoring drug abuse.

Expert consensus on resection of chest wall tumors and chest wall reconstruction.

Chest wall tumors are a relatively uncommon disease in clinical practice. Most of the published studies about chest wall tumors are usually single-center retrospective studies, involving few patients. Therefore, evidences regarding clinical conclusions about chest wall tumors are lacking, and some controversial issues have still to be agreed upon. In January 2019, 73 experts in thoracic surgery, plastic surgery, science, and engineering jointly released the Chinese Expert Consensus on Chest Wall Tumor Resection and Chest Wall Reconstruction (2018 edition). After that, numerous experts put forward new perspectives on some academic issues in this version of the consensus, pointing out the necessity to further discuss the points of contention. Thus, we conducted a survey through the administration of a questionnaire among 85 experts in the world. Consensus has been reached on some major points as follows. (I) Wide excision should be performed for desmoid tumor (DT) of chest wall. After excluding the distant metastasis by multi-disciplinary team, solitary sternal plasmacytoma can be treated with extensive resection and adjuvant radiotherapy. (II) Wide excision with above 2 cm margin distance should be attempted to obtain R0 resection margin for chest wall tumor unless the tumor involves vital organs or structures, including the great vessels, heart, trachea, joints, and spine. (III) For patients with chest wall tumors undergoing unplanned excision (UE) for the first time, it is necessary to carry out wide excision as soon as possible within 1-3 months following the previous surgery. (IV) Current Tumor Node Metastasis staging criteria (American Joint Committee on Cancer) of bone tumor and soft tissue sarcoma are not suitable for chest wall sarcomas. (V) It is necessary to use rigid implants for chest wall reconstruction once the maximum diameter of the chest wall defect exceeds 5 cm in adults and adolescents. (VI) For non-small cell lung cancer (NSCLC) invading the chest wall, wide excision with neoadjuvant and/or adjuvant therapy are recommended for patients with stage T3-4N0-1M0. As clear guidelines are lacking, these consensus statements on controversial issues on chest wall tumors and resection could possibly serve as further guidance in clinical practice during the upcoming years.

Alpha-Methylfentanyl and Beta-Hydroxyfentanyl LC-MS-MS Quantification in Rat Plasma after Long-Term Ethanol Exposure.

Fentanyl and its analogues are highly abused drugs that dominate the illicit drug trade. alpha-Methylfentanyl (A-F) and beta-hydroxyfentanyl (B-F) are two fentanyl analogues that require the development of rapid detection technologies. The current study established and validated a rapid and high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to measure A-F and B-F concentrations in rat plasma following intravenous drug administration (20 µg/kg). Because fentanyl is primarily metabolized by the liver, we evaluated the concentrations of A-F and B-F in vivo in rats, in a control group and a group with liver damage induced by 55 days of oral ethanol gavage (6.5 g/kg, 22.5% v/v). Liquid-liquid extraction and LC-MS-MS operating in the positive ion multiple reaction monitoring mode were used. A C18 column was used, and the mobile phase consisted of 0.1% formic acid aqueous and acetonitrile. The limit of detection was 3 pg/mL (S/N > 5) for A-F and B-F. The calibration curves were linear within the concentration range of 0.01-5 ng/mL (R2 = 0.9991) and 0.005-20 ng/mL (R2 = 0.9999) for A-F and B-F, respectively. Extraction recoveries were 91.3%-97.6% with RSD ≤ 11.2% and 90.5%-94.3% with RSD ≤ 10.5% for A-F and B-F, respectively. Plasma matrix effects were 80.61%-84.58% for A-F and 80.67%-81.33% for B-F with RSD ≤ 13.9%. The validated assay indicated no significant differences in pharmacokinetic parameters (AUC0-t, Cmax and T1/2) derived from the assessment of A-F and B-F plasma concentrations between control and ethanol-exposed rats. This assay, for which the LOD was 3 pg/mL for A-F and B-F may help the forensic science field to determine fentanyl analogue-related causes of death and identify illicit drug tampering.

Plasma YKL-40: a Potential Biomarker for Tumor Invasion in Esophageal Cancer.

BACKGROUND: YKL-40, a chitinase-like glycoprotein has been identified as a candidate tumor marker. The current study evaluated the clinical significance of plasma YKL-40 in esophageal cancer patients. METHODS: We enrolled 127 esophageal cancer patients, 29 healthy controls. Plasma YKL-40 levels were measured through enzyme linked immunosorbent assay. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic efficiency of plasma YKL-40 in esophageal cancer patients. The correlations between plasma YKL-40 and clinicopathological characteristics of esophageal were analyzed. RESULTS: Plasma YKL-40 levels were significantly higher in patients with lymph node metastasis than those that were non-metastatic (p = 0.005). Patients with tumor thrombus formation presented with significantly higher YKL-40 levels than those without thrombus formation (160.3 vs. 74.7 ng/mL, p = 0.012). YKL-40 levels in patients with advanced stage (III and IV) were significantly higher than those in the early stages (I and II, p = 0.016). ROC curve analysis showed that the area under curve was 0.909, and the best diagnostic threshold of YKL-40 for esophageal cancer was 80.6 ng/mL with 68.9% sensitivity and 96.6% specificity. CONCLUSIONS: This study indicated that YKL-40 may be a biomarker for esophageal cancer and potential biomarker for identification of invasive esophageal cancer.

Knockdown of Rap2B Inhibits the Proliferation and Invasion in Hepatocellular Carcinoma Cells.

Rap2B, a member of the Ras family of small GTP-binding proteins, was found to be highly expressed in various human tumors and plays an important role in the development of tumors. However, the function of Rap2B in hepatocellular carcinoma (HCC) remains unclear. Therefore, in this study, we investigated the biological functions of Rap2B in HCC and the potential underlying mechanisms. Our results indicated that Rap2B was highly expressed in HCC tissues and cell lines. Rap2B silencing obviously inhibited the proliferation, migration, and invasion of HCC cells, as well as attenuated xenografted tumor growth in vivo. Furthermore, Rap2B silencing greatly reduced the expression levels of phosphorylated focal adhesion kinase (p-FAK), matrix metalloproteinase-2 (MMP-2), and MMP-9 in HCC cells. In conclusion, our data suggest that Rap2B silencing inhibits the proliferation and invasion in HCC cells. Thus, Rap2B may have potential as a treatment for HCC.

PRSS8 is Downregulated and Suppresses Tumour Growth and Metastases in Hepatocellular Carcinoma.

BACKGROUND: Protease serine 8 (PRSS8), a trypsin-like serine peptidase, has been shown to function as a tumour suppressor in various malignancies. The present study aimed to investigate the expression pattern, prognostic value and the biological role of PRSS8 in human hepatocellular carcinoma (HCC). METHODS: PRSS8 expression in 106 HCC surgical specimens was examined by Real-time polymerase chain reaction (PCR) and immunohistochemistry, and its clinical significance was analysed. The role of PRSS8 in cell proliferation, apoptosis and invasion were examined in vitro and in vivo. RESULTS: PRSS8 mRNA and protein expression were decreased in most HCC tumours from that in matched adjacent non-tumour tissues. Low intratumoral PRSS8 expression was significantly correlated with poor overall survival (OS) in patients with HCC (P = 0.001). PRSS8 expression was an independent prognostic factor for OS (hazard ratio [HR] = 1.704, P = 0.009). Furthermore, restoring PRSS8 expression in high metastatic HCCLM3 cells significantly inhibited cell proliferation and invasion. In contrast, silencing PRSS8 expression in non-metastatic HepG2 cells significantly enhanced cell growth and invasion. Moreover, our in vivo data revealed that attenuated PRSS8 expression in HepG2 cells greatly promoted tumour growth, while overexpression of PRSS8 remarkably inhibited tumour growth in an HCCLM3 xenograft model. Enhanced cell growth and invasion ability mediated by the loss of PRSS8 expression was associated with downregulation of PTEN, Bax and E-cadherin and an upregulation in Bcl-2, MMP9 and N-cadherin. CONCLUSIONS: Our data demonstrate that PRSS8 may serve as a tumour suppressor in HCC progression, and represent a valuable prognostic marker and potential therapeutic target for HCC.

Interplay between menin and K-Ras in regulating lung adenocarcinoma.

MEN1, which encodes the nuclear protein menin, acts as a tumor suppressor in lung cancer and is often inactivated in human primary lung adenocarcinoma. Here, we show that the inactivation of MEN1 is associated with increased DNA methylation at the MEN1 promoter by K-Ras. On one hand, the activated K-Ras up-regulates the expression of DNA methyltransferases and enhances the binding of DNA methyltransferase 1 to the MEN1 promoter, leading to increased DNA methylation at the MEN1 gene in lung cancer cells; on the other hand, menin reduces the level of active Ras-GTP at least partly by preventing GRB2 and SOS1 from binding to Ras, without affecting the expression of GRB2 and SOS1. In human lung adenocarcinoma samples, we further demonstrate that reduced menin expression is associated with the enhanced expression of Ras (p < 0.05). Finally, excision of the Men1 gene markedly accelerates the K-Ras(G12D)-induced tumor formation in the Men1(f/f);K-Ras(G12D/+);Cre ER mouse model. Together, these findings uncover a previously unknown link between activated K-Ras and menin, an important interplay governing tumor activation and suppression in the development of lung cancer.

Direct determination of anabolic steroids in pig urine by a new SPME-GC-MS method.

A new solid phase microextraction (SPME) method coupled with gas chromatography-mass spectrometry (GC-MS) was developed for rapid determination of four anabolic steroids such as 3alpha-hydroxy-5alpha-androstane-17-one (HA), dihydrotestosterone (DHT), androstenedione (AD) and methyltestosterone (MT) in pig urine. SPME was used to extract the four anabolic compounds directly without derivatization. The optimum SPME sampling conditions were based on the home-made carbowax-divinylbenzene (CW-DVB) fiber coating during extraction at 40 degrees C for 50 min with 0.18 g/mL NaCl solution and 750 rpm stirring speed. The linear ranges of the proposed method were in the range of 8-640 pg/mL for HA and DHT and 16-510 pg/mL for AD and MT, respectively. The detection limits (S/N=3) were from 2 to 8 pg/mL for the four anabolic steroids. This SPME method provided very high enrichment factors for the four anabolic steroids, which were 1063-fold and 965-fold for HA and DHT at the concentration of 8 pg/mL and 207-fold and 451-fold for AD and MT at the concentration of 16 pg/mL, respectively. The recoveries ranged from 71.3 to 121%, and the RSDs were lower than 12.9%. The method was sensitive and reliable for determination of trace anabolic steroids in biological samples.

A method by homemade OH/TSO-PMHS fibre solid-phase microextraction coupling with gas chromatography-mass spectrometry for analysis of antiestrogens in biological matrices.

A kind of homemade solid-phase microextraction fibre coupled with gas chromatography-mass spectrometry (GC-MS) was developed for trace analysis of antiestrogens (tamoxifen, cis- and trans-clomiphene) in biological matrices. In this method, derivatization was unnecessary and sample solution could be injected directly after very simple deproteinization operation. The conditions of influencing adsorption of the solid-phase microextraction (SPME) fibre and desorption of the analytes were investigated in details. Matrix effects were studied in different background. Under optimum conditions, the proposed method was further validated by spiking analytes into rabbit liver solutions. Linear ranges of tamoxifen, cis- and trans-clomiphene were 0.02-2.56, 0.08-2.56 and 0.16-2.56 ng mL(-1), respectively. The limits of quantitation were in the range of 0.02-0.16 ng mL(-1). The intra-day accuracy was ranged 96.2-106.2% and precision were in the range of 5.1-8.7%. The extraction recoveries of the antiestrogens in rabbit liver solution were between 73.8% and 113.1%, and R.S.D.s were from 3.6% to 14.1%. The results show that the homemade sol-gel coating is suitable for determination of trace antiestrogens in complex matrices. The proposed approach was proved to be rapid, simple, easy, sensitive and reproducible for trace analysis of antiestrogens in biological matrices.