Simultaneous analysis of 31 anti-impotence compounds potentially illegally added to herbal-based dietary supplements by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

In this paper, an ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF HRMS) method was developed and validated for screening, confirmation and quantitation of 31 anti-impotence compounds potentially illegally added to herbal-based dietary supplements. The analytes were well separated by the mobile phase consisted of 0.1% formic acid solution and acetonitrile with gradient elution at a flow rate of 0.3 mL/min. The MS analysis was operated in positive mode and the mass error of the 31 compounds were below 2.9 ppm. The method validation showed good linearity with coefficients of determination (r2) higher than 0.9973 for all analytes. LODs and LLOQs ranged from 0.005 to 0.50 µg/g or µg /mL and from 0.02 to 1.24 µg /g or µg/mL, respectively. The accuracy was in the range of 86.6% to 113.7%, while the intra-and inter-day precision were in the ranges of 0.9-7.6% and 0.9-11.4%, respectively. The absolute and relative matrix effect were in the range of 65.8-115.6% and 0.6-13.3%. The mean recoveries were in the range of 80.5-116.9%. The stability ranged from 0.4% to 8.5%. Among 200 batches of herbal-based dietary supplements, sildenafil and/or tadalafil were found to be added illegally in two samples, while not very high concentration of icariin was detected in one sample. The Q-TOF mass spectrometry has been proved to be a very powerful and efficient tool for rapid screening of 31 anti-impotence compounds potentially illegally added to herbal-based dietary supplements, ensuring food safety and public health.

MGMT promoter methylation and 1p/19q co-deletion of surgically resected pulmonary carcinoid and large-cell neuroendocrine carcinoma.

BACKGROUND: The response to temozolomide (TMZ) treatment in small-cell lung cancer (SCLC) correlated with O(6)-methylguanine -DNA methyltransferase (MGMT) promoter methylation. 1p/19q co-deletion within oligodendroglioma is a responsive predictor for TMZ. Currently, the status of MGMT promoter methylation and 1p/19q co-deletion in pulmonary carcinoid (PC) and large-cell neuroendocrine carcinoma (LCNEC) is not reported. METHODS: Nine PC [two atypical carcinoids (AC), seven typical carcinoids (TC)] and six LCNEC patients were collected retrospectively. The pyrosequencing and fluorescence in situ hybridization were used to detect the MGMT promoter methylation and 1p/19q co-deletion in surgically resected specimens. Kaplan-Meier analysis was used to assess the rate of disease-free survival (DFS). RESULTS: MGMT promoter methylation was found in two (2/6, 15.3%) LCNEC patients but not in any PC patients. Three (3/6, 50%) 1p and two (2/6, 33.3%) 19q single deletions were found in LCNEC patients. One 1p single deletion was found in AC patients. One (1/7, 14.3%) 1p and two (2/7, 28.6%) 19q single deletions were found in TC patients. After a median follow-up of 38 months, three LCNEC patients developed distant metastasis and one patient died of LCNEC disease. The DFS of PC patients was much longer than LCNEC patients (χ 2 = 7.565, P = 0.006). CONCLUSIONS: MGMT promoter methylation and 1p/19q co-deletion might not be the ideal biomarkers for TMZ treatment in TC/AC patients. Thus, the detection of MGMT promoter methylation and whether it can be used as a medication for TMZ in LCNEC patients necessitates investigation. Furthermore, 1p deletion could be a negative prognostic factor for LCNEC patients.

The Role and Mechanism of Borneol to Open the Blood-Brain Barrier.

BACKGROUND: The blood-brain barrier (BBB) is the greatest challenge in the treatment of intracranial malignant tumors. OBJECTIVE: The aim of this study is to determine the role of borneol in opening the BBB and elucidate the underlying mechanisms. MATERIALS AND METHODS: Twenty Sprague-Dawley (SD) rats were randomized into borneol group intragastrically administered with 10% borneol corn oil (2 mL/kg) and control group. After 30 minutes, 2% Evans blue (4 mL/kg) was injected. Thirty minutes later, brain tissue was analyzed using the Evans blue standard curve. Another 40 SD rats were randomized into high-, medium-, and low-dose borneol groups and a control group. Each rat in the experimental groups was intragastrically administered with 10% borneol corn oil (2 mL/kg, 1.25 mL/kg, and 0.5 mL/kg, respectively). The control group was injected with corn oil of 1.25 mL/kg. After 30 minutes, the rats were killed, and the brain tissues were collected. The expression of occludin, occludens-1, nitric oxide synthase, P-glycoprotein, and intercellular cell adhesion molecule-1 (ICAM-1) was detected by immunohistochemy. RESULTS: The concentration of Evans blue in the borneol group was higher than in the control group ( P < .05). The mean density of ICAM-1 expression was higher in the experimental group than in the control group ( P < .05). In contrast, significant differences of positive area and total density of ICAM-1 were shown only between the high-dose group and the control group ( P < .05). CONCLUSION: Borneol can open the BBB, which might be related with the increased expression of ICAM-1.

Application of ultra-high-performance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry for identification, confirmation and quantitation of illegal adulterated weight-loss drugs in plant dietary supplements.

In this paper, an ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap HRMS) method was developed and validated for identification, confirmation and quantitation of illegal adulterated weight-loss drugs in plant dietary supplements. 13 wt-loss drugs were well separated by the gradient elution of 10mmol/L ammonium acetate - 0.05% formic acid H2O and acetonitrile at a flow rate of 0.2mL/min within 12min. The MS analysis was operated under the positive ion and in full MS/dd-MS2 (data-dependent MS2) mode. The full MS scan with resolution at 60 000 FWHM and narrow mass windows at 5ppm acquired data for identification and quantitation, and dd-MS2 scan with resolution at 15 000 FWHM obtained product ions for confirmation. The method validation showed good linearity with coefficients of determination (r2) higher than 0.9951 for all analytes. Meantime, all the LOD and LLOQ values were in the respective range of 0.3-2 and 1-9ng/g. The accuracy, intra- and inter-day precision were in the ranges of -1.7 to 3.4%, 1.7-5.0% and 1.9-4.4%, respectively. The mean recoveries ranged from 85.4 to 107.1%, while the absolute and relative matrix effect were in the corresponding range of 98.2-108.6% and 2.6-8.7%. Among 120 batches of weight loss plant dietary supplements, sibutramine and fluoxertine or both were positive in 29 samples. In general, LTQ-Orbitrap HRMS technology was a powerful tool for the analysis of illegal ingredients in dietary supplements.

PIK3CA Mutations in Resected Small Cell Lung Cancer.

BACKGROUND: Despite advances in chemotherapy and radiotherapy in recent decades, the prognosis for small cell lung cancer (SCLC) patients is still poor. Targeted therapies in SCLC must be applied systemically to target not only the primary tumor but also metastases. The phosphatidylinositol 3-kinase (PI3K)/AKT pathways play a key regulatory function in the survival, proliferation, energy metabolism and cellular architecture advantages of malignant cells. The phosphatidylinositol 3-kinase catalytic α (PIK3CA) gene, which encodes the p110α catalytic subunit, plays a key role in the activation of AKT downstream signaling and mammary tumor progression. More than 75% of PIK3CA mutations are clustered in the helical (exon 9) and catalytic domains (exon 20). There have been very few studies reporting the PIK3CA mutations status of patients with SCLC who have undergone surgical treatment in mainland China. OBJECTIVES: The aim of the study was to investigate the PIK3CA mutation in SCLC. MATERIAL AND METHODS: Reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing technology was used to detect the PIK3CA mutation in 14 cases of retrospectively collected SCLC patients who underwent surgical treatment at Zhejiang Cancer Hospital, Hangzhou, PRC, between 2002 and 2010. RESULTS: The research revealed no mutations in exons 9 and 20 of the PIK3CA gene. A nucleotide alteration of A1634C (E545A) of exon 9 turned out to be a pseudogene-positive, because the mutation disappeared when near-duplicate detection was employed. CONCLUSIONS: The incidence of PIK3CA mutation is low in SCLC patients, and the pseudogene-positive alteration of A1634C is prone to occur in exon 9 of PIK3CA mutations in human cancers.

Characterization of a novel process-related impurity in commercial bendazac lysine eye drops by LC-ESI-QTOF/MS/MS and NMR.

A simple and sensitive method was developed for characterization of two impurities from commercial bendazac lysine eye drops, by using liquid chromatography combined with electrospray ionization and QTOF mass analyzer (LC-ESI-QTOF MS/MS). A known impurity was characterized rapidly by comparison with the retention time and MS/MS data of the reference standard. Based on high resolution mass spectrometry (HRMS) and the nuclear magnetic resonance (NMR), a new impurity, purified by preparative HPLC, was identified as 2-(1,5-dibenzyl-1H-indazol-3-yloxy)acetic acid. In addition, a plausible mechanism for the formation of the impurities was also proposed.

Nanoplasmonic biosensor: coupling electrochemistry to localized surface plasmon resonance spectroscopy on nanocup arrays.

The nanoscale Lycurgus cup arrays were hybrid structures of nanocups and nanoparticles with ultrasensitivity to refractive index change. In this study, an electrochemical localized surface plasmon resonance (LSPR) sensor was developed by coupling electrochemistry to LSPR spectroscopy measurement on the nanoscale cup arrays (nanoCA). Based on the combination of electrochemistry and LSPR measurement, the electrochemical LSPR on nanoCA was observed with significant resonance wavelength shifts in electrochemical modulation. The synchronous implementation of cyclic voltammetry and optical transmission spectrum can be used to obtain multiply sensing information and investigate the enhancement for LSPR from electrochemical scanning. The electrochemical enhanced LSPR was utilized as biosensor to detect biomolecules. The electrochemical LSPR biosensor with synchronous electrochemical and optical implement showed higher sensitivity than that of conventional optical LSPR measurement. Detecting with multi-transducer parameters and high sensitivity, the electrochemical LSPR provided a promising approach for chemical and biological detection.

Identifying EGFR mutations from SCLC patient plasma by mutant-enriched liquidchip technology.

BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) such as erlotinib and gefitinib are targeted drugs for the kinase domain of EGFR. They are widely used for the treatment of non-small cell lung cancer (NSCLC). The EGFR exon 19 deletion mutation and the L858R mutation in exon 21 comprise approximately 90% of the somatic mutations in NSCLC patients that respond to EGFR TKI. Several recent studies have also reported that small cell lung cancer (SCLC) patients with EGFR mutations responded to gefitinib. Further study, however, has been limited due to the difficulty obtaining tumor specimens from SCLC patients. OBJECTIVES: The aim of this study was to explore the EGFR mutation status in SCLC patients by plasma analysis. MATERIAL AND METHODS: Plasma samples from SCLC patients were collected for mutant-enriched liquidchip (MEL) analysis to identify the EGFR mutations in exon 19 and 21. RESULTS: The exon 19 deletion mutation was detected in one out of 35 patients (a female non-smoker). No exon 21 mutations were found. CONCLUSIONS: A prevalence of EGFR mutations in SCLC is rare, and occurs most frequently in females and nonsmokers.

[Determination of 7-ethyl-10-hydroxycamptothecin in microdialysates from rat brain with LC-MS/MS].

OBJECTIVE: To establish a method for determination of 7-ethyl-10-hydroxycamptothecin (SN-38) in microdialysates from rat brain. METHODS: The concentrations of SN-38 were measured by LC-MS/MS method with Agilent Eclipse Plus C18 (2.1 mm ×100 mm, 1.8 µm) reversed phase column using acetonitrile-0.1% methanoic acid as mobile phase with gradient elution at a flow rate of 0.3 ml/min and temperature at 35 degree. Multiple reaction monitoring using the precursor to product ion combinations of m/z 393.1→349.1 was performed to detect SN-38 in microdialysates from rat brain. RESULTS: Blank microdialysate had non-interference. The method was linear over the concentration range of 0.1015-1015 ng/ml (r=0.9995); and the lower limit of quantification (LOQ) was 0.1015 ng/ml. The recovery of assay for SN-38 ranged from 97.54%-100.60%. The intra- and inter-day precision and stability were both well. The concentrations of SN-38 in brain microdialysates presented pharmacokinetics process and achieved the peak after 220 min. CONCLUSION: The fully validated LC-MS/MS analytical method has high specificity and sensibility, which can be used effectively to analyze SN-38 in microdialysates from rat brain.

Expression and mutation of the c-kit gene and correlation with prognosis of small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive and lethal type of cancer in humans. SCLC is sensitive to chemotherapy and radiotherapy, but long-term survival is low and the majority of patients eventually develop progressive disease. With the success of imatinib mesylate in the treatment of gastrointestinal stromal tumors expressing c-kit, its use in SCLC serves as a novel molecular therapeutic approach. The activity of imatinib mesylate is correlated with the mutation of c-kit gene exons 9 and 11 in gastrointestinal stromal tumors. The incidence of epidermal growth factor receptor mutation in non-small cell lung cancer is higher in China than in the United States of America and European countries. There may be also differences in the incidence of c-kit mutation between China and European countries. At present, no study examining imatinib mesylate treatment for SCLC in China is available. To investigate the expression and mutation of c-kit and the correlation with prognosis of SCLC in China, immunohistochemistry was used to detect the expression of c-kit, and a pyrosequencing assay was used to detect mutations in c-kit exons 9 and 11 of 36 SCLC patients who received surgical treatment at the Zhejiang Cancer Hospital, Hangzhou, China, between 1998 and 2010. All 36 patients were followed up to analyze the correlation between prognosis and expression and mutation of c-kit. The incidence of c-kit-positive expression was 83.3%, including 25.0% weak staining, 22.2% moderate staining and 36.1% strong staining. The overall survival of patients with c-kit strong staining was shorter compared to patients with c-kit not strong staining. No mutation in c-kit exons 9 and 11 was detected. In conclusion, the findings showed that the expression of c-kit is high, and strong staining is a prognostic factor for worse survival.

Mutation status of epidermal growth factor receptor and clinical features of patients with combined small cell lung cancer who received surgical treatment.

The mutation status of epidermal growth factor receptor (EGFR) is correlated with the response of tumors to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC), suggesting its usefulness as a biomarker in NSCLC. The incidence of EGFR mutation in NSCLC is higher in China than in the United States and European countries. There have been some case reports concerning cases of gefitinib-responsive small cell lung cancer (SCLC) with EGFR mutations. However, few large studies concerning the mutation status of SCLC patients have been performed. We detected EGFR mutations in exons 19 and 21 of 40 SCLC patients, three of whom had combined SCLC, from the Zhejiang Cancer Hospital using xTAG technology. Only two patients with combined SCLC had an EGFR mutation in exon 19. To determine the EGFR mutation status and clinical features of combined SCLC, we retrospectively analyzed the clinical features of seven patients with combined SCLC who had undergone surgical treatment in Zhejiang Cancer Hospital between 2007 and 2010. EGFR mutations in exons 19 and 21 were detected using the pyrosequencing assay. Of the seven patients with combined SCLCs, 71.4% were male, 71.4% were heavy smokers, most were over 60 years old and 71.4% of the cases were combined adenocarcinoma. Chemotherapy treatment and tumor stage were correlated with survival time. Of the seven cases, one had a mutation in exon 19 of EGFR in both the conventional SCLC and SCLC combined adenocarcinoma components. Combined SCLC commonly occurs in patients who are heavy smokers, male and over 60 years old, and most of the combined type cases are adenocarcinoma. The treatment of combined SCLC may be applied to cases of conventional SCLC. EGFR mutations may therefore occur in combined SCLCs, especially in SCLC combined adenocarcinoma in China.

Brain transport of neurotoxin-I with PLA nanoparticles through intranasal administration in rats: a microdialysis study.

The purpose of this study was to encapsulate neurotoxin-I (NT-I) within polylactic acid (PLA) nanoparticles (NPs) and to evaluate their transport into the brain after intranasal administration (i.n.) using a microdialysis sampling technique. NT-I-NPs (NT-I radiolabeled with sodium [(125)I]iodide) were prepared and characterized. Then, NT-I-NPs were administered i.n. or i.v. to rats and the radioactivities in the olfactory bulbs were monitored for up to 240 min. The nanoparticles prepared were spherical with a homogenous size distribution. The mean particle size, zeta potential and entrapment efficiency were -28.6+/-2.3 mV, 65 nm and 35.5+/-2.8%, respectively. The brain transport results showed that the time to reach the peak level (T(max)) of NT-I-NPs (i.n.) was 65 min, shorter than NT-I-NPs (i.v.) (95 min) or NT-I (i.v.) (145 min). The concentration at peak level (C(max)) and the total area under the concentration-time curves from zero to 4 h (AUC(0-4 h)) of each group followed the following order: NT-I-NPs (i.n.)>NT-I-NPs (i.v.)>NT-I (i.v.). The corresponding absolute bioavailabilities (Fabs) of NT-I-NPs (i.n.) were about 160%, 196% with NT-I-NPs (i.v.) and NT-I (i.v.) as reference preparations, respectively. The brain delivery of NT-I could be enhanced with PLA nanoparticles either through i.n. or i.v. administration. Furthermore, the enhancement was more significant for i.n. than for i.v. administration. Nanoparticles as carriers would be a potential way to improve the brain transport for centrally active peptides.

[Brain delivery of neurotoxin-I-loaded nanoparticles through intranasal administration].

The purpose of this paper is to encapsulate neurotoxin-I (NT-I), a kind of analgesic peptide, into polylactic acid (PLA) nanoparticles (NPs) and to evaluate their transport into the brain after intranasal administration (in) by use of microdialysis sampling technique developed in our laboratory recently. NT-I-NPs (NT-Iradiolabeled with sodium 125I-Iodide) were prepared by a double emulsification solvent evaporation method, and were characterized in terms of surface morphology, particle size distribution, zeta potential and entrapment efficiency. Then, NT-I-NPs were administered intranasally or intravenously to rats and the radioactivities in periaqueductal gray (PAG) were monitored up to 240 min utilizing the microdialysis sampling technique. Nanoparticles prepared were spherical with homogenous size distribution. Their mean particle size and zeta potential measured were (65.3 +/- 10.8) nm and (-28.6 +/- 2.3) mV, respectively. The entrapment efficiency of NT-Iencapsulated into nanoparticles was (35.5 +/- 2.8)%. The brain transport results showed that the time to peak level (Tmax) of NT-I-NPs (in) was (65 +/- 10) min approximately, apparently shorter compared with NT-I-NPs [iv, (95 +/- 10) min] or NT-I [iv, (145 +/- 10) min]. The concentration to peak level (Cmax) and the area under the curves from zero to 4 h (AUC0-4h) of each group followed this order: NT-I-NPs (in) > NT-I-NPs (iv) > NT-I (iv). With nanoparticles as carriers and administered intranasally could be a potential way for centrally active peptides to improve their brain transport. Microdialysis is quite a good technique for the study of drug delivery to the brain.

On-line microdialysis coupled to analytical systems.

In vivo sampling of interstitial fluid using microdialysis (MD) fibers has become a standard and accepted procedure. The resulting small volume samples, often with low concentrations of the analyte of interest, present a particular challenge to analytical methods. Rapid developments of analytical techniques with high sensitivity accelerate their combination with MD. On-line MD-based analytical systems are receiving increasing attention because they can provide near real-time data prior to the off-line system. The purpose of this review is to provide information for on-line MD-analytical systems. Special emphasis has been given to the main progression on methodologies of each method during recent years. The advantages, limitations, and adaptivity are also discussed. These methods include on-line MD-liquid chromatography, on-line MD-capillary electrophoresis, on-line MD-mass spectrometry, and on-line MD-biosensor.

Delivery of 125I-cobrotoxin after intranasal administration to the brain: a microdialysis study in freely moving rats.

In order to determine the contribution of intranasal (i.n.) administration to the uptake of large molecular weight (MW) substances into central nervous system (CNS), concentration in brain of the centrally acting polypeptide cobrotoxin (NT-I) versus time profiles were studied using dual-probe microdialysis in awake free-moving rats. NT-I, radiolabeled with sodium (125)I-Iodide ((125)I-NT-I), was administered at the dose of 105 microg/kg intravenously and intranasally in the same set of rat (n=15). The (125)I-NT-Inasal preparations were formulated with borneol/menthol eutectic mixture (+BMEM) as an absorption enhancer and without (-BMEM). After application, the dialysates sampled simultaneously from olfactory bulb and cerebellar nuclei were measured in a gamma-counter for radioactivity. The real concentrations of NT-I were recalculated by in vivo recoveries of microdialysis probes. The results showed that the area under the curve (AUC) value in cerebellar nuclei (2283.51+/-34.54 min ng/ml) following i.n. administration (+BMEM) was significantly larger than those (AUC(olfactory)=1141.92+/-26.42 min ng/ml; AUC(cerebellar)=1364.62+/-19.35 min ng/ml) after intravenous (i.v.) bolus, respectively. A prolonged time values to peak concentrations after i.n. application (+BMEM) were observed compared with those following i.v. administration. Also, following i.n. application (+BMEM) the measured time value to peak concentration in cerebellar nuclei (85 min) was statistically longer than that in olfactory bulb (75 min), which could be plausibly an indication for NT-I delivery into brain via nose-brain pathway in the presence of absorption enhancer. i.n. administration (-BMEM) had little or no ability of NT-I delivering into brain. In conclusion, i.n. administration (+BMEM) significantly enhanced brain transport of NT-I with uneven distribution in discrete regions of brain compared with i.v. administration. Additionally, multi-probe microdialysis technique should be considerably valuable in brain delivery studies.

Intracerebral microdialysis technique and its application on brain pharmacokinetic-pharmacodynamic study.

Intracerebral microdialysis (IC-MD) has been developed as a well-validated and powerful technique for decades. As a practical sampling tool, it can gain the continuous dialysates of endogenous and exogenous substances in extracellular fluid (ECF) of awake freely moving animals. Also, variform IC-MD probes (IC-MDPs) have grown more exquisite. The implantation of the IC-MDP in certain tissue of brain allows monitor drug distribution and measure drug and corresponding neurotransmitters levels in brain ECF after administration for brain pharmacokinetic-pharmacodynamic (B-PK-PD) study. So it is suitable for IC-MD to B-PK-PD study (IC-MD/B-PK-PD). The performance of IC-MD/B-PK-PD can not only elevate the degree of precision and accuracy of experimental data, minimize the individual difference by reduced number of animals, but also give important information for the prediction and optimization of drug effective dose in preclinical study. In this review, we have discussed various IC-MD/B-PK-PD studies of analgesic, antiepileptic and antidepressant drug. The role of IC-MD/B-PK-PD in confirming and assessing the drug effect before clinic trials is highlighted.