Quantification of free polyamines and their metabolites in biofluids and liver tissue by UHPLC-MS/MS: application to identify the potential biomarkers of hepatocellular carcinoma.

Polyamines and their N-acetylated metabolites are potential biomarkers in the diagnosis and therapeutic evaluation of cancer. Thus, we present here an ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of 6 free, 3 monoacetylated, and 2 diacetylated polyamines without derivatization. The major improvement of this method is the use of 0.2 % perfluoroheptanoic acid methanol in the pretreatment step to achieve protein precipitation and 0.0125 % perfluoroheptanoic acid in the mobile phase to achieve analyte separation within 9 min. The established analytical method was validated with plasma, urine, and liver tissue and applied to determine plasma, urine, and liver tissue samples from healthy rats, hepatocellular carcinoma rats, and administrated rats successfully. Results indicated free polyamines such as putrescine mainly existed in liver tissue but more polar N-acetylated metabolites such as N (1),N (12)-diacetylspermine seemed to exist in biological fluid. After carcinogenesis, the levels of polyamines were increased, but the elevated levels of polyamines and their metabolites tended to decrease when administrated with anticancer drug. The method provided a more versatile manner for clinical application in the diagnosis and therapeutic evaluation of hepatocellular carcinoma.

Ultra-fast LC-ESI-MS/MS method for the simultaneous determination of six highly toxic Aconitum alkaloids from Aconiti kusnezoffii radix in rat plasma and its application to a pharmacokinetic study.

A fast, sensitive, and efficient ultra-fast LC-ESI-MS/MS method was developed for the simultaneous quantitation of six highly toxic Aconitum alkaloids, that is, aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, in rat plasma after oral administration of crude ethanol extracts from Aconiti kusnezoffii radix by ultrasonic extraction, reflux extraction for 1 h, and reflux extraction for 3 h, respectively. The separation of six Aconitum alkaloids and aminopyrine (internal standard) was performed on an InertSustain® C18 column, and the quantification of the analytes was performed on a 4000Q ultra-fast LC-MS/MS system with turbo ion spray source in the positive ion and multiple-reaction monitoring mode. Absolute recoveries ranged within 65.06-85.1% for plasma samples. The intra- and interday precision and accuracy of analytes were satisfactory. The methods were validated with sensitivity reaching the lower LOQ for aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, which were 0.025, 0.025, 0.050, 0.025, 0.025, and 0.100 ng/mL, respectively. The method was successfully applied to a pharmacokinetic study of six Aconitum alkaloids in rat plasma after oral administration of crude ethanol extracts from the raw root of Aconitum kusnezoffii Reichb. by three different extraction processes.

Simultaneous quantitation of polygalaxanthone III and four ginsenosides by ultra-fast liquid chromatography with tandem mass spectrometry in rat and beagle dog plasma after oral administration of Kai-Xin-San: application to a comparative pharmacokinetic study.

A fast, selective, and quantitative ultra-fast liquid chromatography with tandem mass spectrometry method has been developed and validated for the simultaneous quantitation of polygalaxanthone III, ginsenoside Rb1, ginsenoside Rd, ginsenoside Re, and ginsenoside Rg1 in the plasma of rat and beagle dog after oral administration of Kai-Xin-San. After addition of the internal standard, salidroside, the plasma samples were extracted by liquid-liquid extraction and separated on a Venusil MP C18 column with methanol/0.01% acetic acid water as mobile phase. The tandem mass spectrometric detection was performed in the multiple reaction monitoring with turbo ion spray source in a switching ionization mode. The method was examined, and found to be precise and accurate with the linearity range of the compounds. The intra- and interday precision and accuracy of the analytes were well within acceptance criteria (±15%). The mean extraction recoveries of analytes and internal standard were all >75.0%. The validated method has been successfully applied to comparing pharmacokinetic profiles of analytes in rat and beagle dog plasma. The results indicated that no significant differences were observed in pharmacokinetic parameters of ginsenoside Rg1, while the others had significant differences, which may due to the different mechanisms of absorption and metabolism.

Sophora flavescens-Astragalus mongholicus herb pair in the progression of hepatitis, cirrhosis, and hepatocellular carcinoma: a possible mechanisms and relevant therapeutic substances.

Background: Both Sophora flavescens (SF) and Astragalus mongholicus (AM) are known for their anti-inflammatory, antifibrotic, and anticancer activities. However, the efficacy, multi-target mechanisms, and therapeutic substances of SF-AM herb pair on the progression of hepatitis-cirrhosis-hepatocellular carcinoma hepatocellular carcinoma (HCC) remain unclear. Purpose: To investigate the efficacy, mechanisms, and potential therapeutic substances of SF-AM herb pair in the progression of hepatitis-cirrhosis-HCC. Methods: Firstly, diethylnitrosamine was used to establish the hepatitis-cirrhosis-HCC model. HE staining and non-targeted metabolomics were used to evaluate the efficacy of SF-AM herb pair. Subsequently, the absorbed components of SF-AM herb pair in the plasma of rats were determined through HPLC-Q-TOF-MS/MS analysis. Flow cytometry, Western blot, and qRT-PCR were then employed to assess CD4+ and CD8+ T lymphocytes, PI3K/Akt signaling pathway-related proteins, and their corresponding mRNAs. Simultaneously, the efficacy and mechanism of SF-AM herb pair on HCC were confirmed by in vitro experiments. Finally, Pearson correlation analysis was performed between pharmacodynamic indicators and in vivo components to identify the potential therapeutic substances of SF-AM herb pair. Results: SF-AM herb pair can alleviate the pathological damage and reverse metabolic abnormalities in hepatitis, cirrhosis, and HCC rats, particularly during the hepatitis and cirrhosis stages. Pharmacological researches have demonstrated that SF-AM herb pair can increase the proportion of CD8+ T lymphocytes, inhibit the expression of PI3K, Akt, p-Akt, NF-κB p65, NF-κB pp65, and Bcl-2, as well as increase the expression of IκBα, Bax, and cleaved caspase-3. These findings suggest that SF-AM herb pair has the ability to enhance immunity, anti-inflammation and promote apoptosis. Cell experiments have shown that SF-AM herb pair can inhibit the proliferation of HepG2 cell and regulate the PI3K/Akt signaling pathway. Moreover, 23 absorbed prototypical components and 53 metabolites of SF-AM herb pair were identified at different stages of HCC rats. Pearson correlation analysis revealed that matrine, cytisine, wogonoside, and isoastragaloside are potential therapeutic substances in SF-AM herb pair for the prevention and treatment of hepatitis, cirrhosis, and HCC. Conclusion: In summary, this study revealed the efficacy, mechanisms, and potential therapeutic substances of SF-AM herb pair in the hepatitis-cirrhosis-HCC axis and provided a reference for its clinical application.

Accurate determination for lipidomics based on LC-tandem-MS parameters modeling, prediction, and database: Monitoring the progression of hepatocellular carcinoma.

The unavailability of adequate lipid reference standards is a major challenge for accurate quantitative analysis of lipidomics. Based on the discovery of regularity and predictability for lipids in chromatography-mass spectrometry behaviors, "target compound-structure correlation-analytical parameter database" protocol and "modeling-prediction" strategy were carried out to calculate the relative coefficients of analytical parameters within each subclass. Then the relevant LC-tandem-MS parameters of unknown lipids were predicted and a quantification parameter database for 4081 lipids was established and validated. Reference standards-independent accurate determination for lipidomics was achieved with the parameter's database and applied to monitor the change of lipid metabolism in the plasma of whole course of health-hepatitis-cirrhosis-hepatocellular carcinoma (HCC). Combined Student's t test, orthogonal partial least squares discrimination analysis (OPLS-DA) and binary logistic regression-ROC analysis, lipid biomarkers for differentiating health from each disease and differentiating different stages of disease were identified and the pathogenesis of HCC was preliminarily clarified. The established methodology would shed light on comprehensive and accurate quantitative lipidomics and exploring the pathomechanism and potential therapeutic targets of HCC.

A new oleanane type pentacyclic triterpenoid saponin from the husks of xanthoceras sorbifolium bunge and its neuroprotection on PC12 cells injury induced by Aß25-35.

A new triterpenoid saponin (1), 3-O-[ß-D-glucopyranosyl(1→6)]-(2'-angeloyl)-ß-D-glucopyranosyl-28-O-ß-D-glucopyranosyl(1→6)[α-L-rhamnopyranosyl(1→2)-ß-D-glucopyranosyl]-21-O-acetyl-16-deoxybarringtogenol C, together with four known saponins (2 ∼ 5) were isolated from the husks of Xanthoceras sorbifolium Bunge. Their structures were characterized by HR-ESI-MS, 1D-NMR, 2D-NMR spectra and chemical methods. Compound 1 exhibited excellent neuroprotection on PC12 cells injury induced by Aß25-35 at the doses of 150 µmol/L and 200 µmol/L. The cell viabilities were (76.18 ± 2.09) % and (76.02 ± 3.20) %, respectively.[Formula: see text].

Quality control of Semen Ziziphi Spinosae standard decoction based on determination of multi-components using TOF-MS/MS and UPLC-PDA technology.

A sensitive, fast and comprehensive method for the quality assessment of Semen Ziziphi Spinosae (SZS) standard decoction with characterization of its chemical components was developed and validated. UPLC-Q/TOF-MS/MS system was used to identify thirty-six chemical components of SZS standard decoction which included nucleosides, phenolic acids, alkaloids, and flavonoids. Furthermore, a UPLC-PDA method was validated to simultaneously determine adenosine, protocatechuic acid, magnoflorine, catechin, protocatechin, vicenin II, spinosin, kaempferol-3-rutinoside, and 6'''-feruloylspinosin which represent four species of characteristic compounds. The qualitative method had been validated according to Chinese Pharmacopoeia (2015 edition) in terms of lineary, repeatability, recovery and stability for all analytes, with the results showing good precision, accuracy and stability. In conclusion, the method using UPLC combined with MS and PDA provided a novel way for the standardization and identification of SZS standard decoction, and also offered a basis for qualitative analysis and quality assessment of the preparations for SZS standard decoction.

Plasma N-acetylputrescine, cadaverine and 1,3-diaminopropane: potential biomarkers of lung cancer used to evaluate the efficacy of anticancer drugs.

Polyamines have been widely investigated as potential biomarkers for various types of cancers, including lung cancer, which is one of the most common causes of death from cancer worldwide. This study was carried out to evaluate the value of polyamines that serve as early diagnostic and cancer progression markers as well as drug evaluation for lung cancer (squamous cell carcinoma of lung, SCCL). SCCL was induced in Wistar rats by intratracheal instillation of 3-methylcholanthrene and treated with three different anti-cancer drugs, Aidi injections, fluorouracil, and a combination of them. After carcinogenesis for 28, 70 and 98 days and therapy for 28 and 56 days, the polyamine levels in plasma of SCCL, healthy and treated rats were determined using a UHPLC-MS/MS assay base on the means of targeted metabolomics. Results showed that increased N-acetylputrescine, cadaverine and 1,3-diaminopropane levels were associated with progression of SCCL. The levels of cadaverine and 1,3-diaminopropane returned to normal after administration of the three different kinds of anticancer drug. In addition, the suitability of using N-acetylputrescine, cadaverine and 1,3-diaminopropane as biomarkers was confirmed by PLS-DA and ROC analysis. It can provide an innovative and effective way for the clinical diagnosis, prevention and treatment of lung cancer, and stimulate a theoretical basis for the design and development of new anticancer drugs. At the same time, this increased the clinical options for polyamines as cancer biomarkers.

A fast, sensitive, and high throughput method for the determination of esomeprazole in dog plasma by UHPLC-MS/MS: Application to formulation development of the compound preparation of esomeprazole.

To investigate deeply into the preclinical pharmacokinetics and prescription design of esomeprazole, a sensitive, high throughput and robust UHPLC-MS/MS method had been developed and fully validated for the analysis of esomeprazole in dog plasma. Esomeprazole and diazepam (IS) were fast extracted from plasma by alkalified organic solvent, and separated on MP-C18 column with methanol and 0.1% formic acid. The quantification of esomeprazole and IS had been achieved using fragmentation transitions of m/z 346.1→198.1 and m/z 285.0→193.2 in MRM detection under positive ESI mode. The concentration of esomeprazole in dog plasma was linear with the range of 3.75-500ng/mL. The precisions of intra- and inter-day were no more than 11.6%, while the accuracies were all within ±9.7% of the nominal values. The recovery was no more than 77.06%, and the matrix effect, stability, dilution integrity tests were all satisfied the currently criterion. Then the method was successfully performed to evaluating pharmacokinetics of esomeprazole and optimizing the prescription of modified esomeprazole with varied addition of sodium bicarbonate. Consequently, a pharmacokinetic study of three doses esomeprazole with the optimized addition of sodium bicarbonate in dogs has been successfully researched for the first time. It could be a promising approach to improve the stabilization of acid-labile esomeprazole and would provide a useful reference for the formulation design of esomeprazole.

Quantitation of eleven active compounds of Aidi injection in rat plasma and its application to comparative pharmacokinetic study.

Aidi injection has been widely used for the treatment of colorectal cancer. The purpose of this study was to develop a sensitive and reliable method for simultaneous quantitation of 11 main active ingredients in Aidi injection and to compare the pharmacokinetics of these ingredients in normal and colorectal model cancer rats after tail vein injection. After being extracted by isopropanol-ethyl acetate (1:1, v/v), the plasma samples were analyzed with domperidone as internal standard. Then the analytes were separated on a Venusil MP C18 column with 0.15% formic acid and methanol. The detection was performed on HPLC-MS/MS system with turbo ion spray source in the positive ion and multiple reaction-monitoring mode. The assay was shown to be linear over the range of 0.004-4.0µgmL(-1) of syringin B, astragaloside II and isofraxidin; 0.01-10.0µgmL(-1) of calycosin-7-O-ß-d-glucoside and astragaloside IV; 0.02-20.0µgmL(-1) of ginsenoside Rg1, Rb1, Rc and Rd; 0.04-40.0µgmL(-1) of syringin E; 0.06-60.0µgmL(-1) of ginsenoside Re. And the validated method has been successfully applied to compare pharmacokinetic profiles of the 11 ingredients in plasma. The pharmacokinetic results showed here were significant differences in pharmacokinetic parameters for eight analytes between two groups after injection, while no significant differences for astragaloside II, astragaloside IV and ginsenoside Rc. The present study has the advantages of short analysis time and easy sample preparation, which could more comprehensively reflect the quality of Aidi injection in single run. The method proposed could be of great use for pharmacokinetics, bioavailability or bioequivalence studies of Aidi injection in biological samples.

Determination of catecholamines and their metabolites in rat urine by ultra-performance liquid chromatography-tandem mass spectrometry for the study of identifying potential markers for Alzheimer's disease.

In order to investigate the potential links between catecholamines (CAs) and Alzheimer's disease (AD), rapid and sensitive ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) methods in different ionization modes for the quantification of 14 CAs and their metabolites in rat urine without derivatization or complex sample pre-treatments were developed. After addition of the internal standard, isoproterenol, the urine samples were extracted by protein precipitation and separated on an Inertsil ODS-EP column (Shimadzu, Japan) at a flow of 1.0 ml min(-1). Tandem mass spectrometric detection was performed on a 4000Q UPLC-MS/MS in the multiple reaction monitoring mode with turbo ion spray source. Tyrosine, dopamine, noradrenaline, epinephrine, 3-methoxytyramine, normetanephrine and metanephrine were determined in positive mode, while 3,4-dihyroxy-L-phenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid, DL-3,4-dihydroxymandelic acid, DL-3,4-dihydroxyphenyl glycol, homovanillic acid, DL-4-hydroxy-3-methoxymandelic acid and 4-hydroxy-3-methoxy-phenylglycol were determined in negative mode. The methods were examined and were found to be precise and accurate within the linearity range of the assays. The intra-day and inter-day precision and accuracy of the analytes were well within acceptance criteria (±15%). The mean extraction recoveries of analytes and internal standard were all more than 60%. The validated methods have been successfully applied to compare CAs profiles in normal and AD rats. The results indicated the urine levels of DL-3,4-dihydroxyphenyl glycol and 4-hydroxy-3-methoxy-phenylglycol in AD rats were significantly higher than those in the normal group, and the other CAs have an opposite performance. These may attribute to the difference of some enzyme activity between rats with AD and normal. Furthermore, this may be helpful in clinical diagnostics and monitor the efficacy of AD treatment.

Enrichment and purification of six Aconitum alkaloids from Aconiti kusnezoffii radix by macroporous resins and quantification by HPLC-MS.

Aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine are six main Aconitum alkaloids from traditional Chinese medicine, Aconiti kusnezoffii radix, which possess highly bioactive as well as highly toxic character for medicinal use. In the present study, for the purpose of better utilizing the toxic herbal material, the performance characteristics of NKA-II, D101, X-5, AB-8, S-8, HPD722 and HPD750 macroporous resins for the enrichment and purification of these six Aconitum alkaloids were critically evaluated. Results showed that NKA-II offered the best adsorption and desorption capacities for six Aconitum alkaloids among the seven macroporous resins tested, which were affected significantly by the pH value. Subsequently, dynamic adsorption and desorption experiments had been carried out with the column packed by NKA-II resin to optimize the separation process of six Aconitum alkaloids. After one run treatment with NKA-II resin, the content of total six Aconitum alkaloids were increased from 5.87% to 60.3%, the recovery was 75.8%. Meanwhile, a validated HPLC-MS method had been developed to qualitative and quantitative these six Aconitum alkaloids. This method would provide scientific references to the large-scale production of six Aconitum alkaloids from Aconiti kusnezoffii radix or other plants and might also expand the secure application of these highly toxic components for pharmacy.

A UFLC-MS/MS method with a switching ionization mode for simultaneous quantitation of polygalaxanthone III, four ginsenosides and tumulosic acid in rat plasma: application to a comparative pharmacokinetic study in normal and Alzheimer's disease rats.

A fast, sensitive and reliable ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method has been developed and validated for simultaneous quantitation of polygalaxanthone III (POL), ginsenoside Rb1 (GRb1), ginsenoside Rd (GRd), ginsenoside Re (GRe), ginsenoside Rg1 (GRg1) and tumulosic acid (TUM) in rat plasma after oral administration of Kai-Xin-San, which plays an important role for the treatment of Alzheimer's disease (AD). The plasma samples were extracted by liquid-liquid extraction using ethyl acetate-isopropanol (1:1, v/v) with salidrdoside as internal standard (IS). Good chromatographic separation was achieved using gradient elution with the mobile phase consisting of methanol and 0.01% acetic acid in water. The tandem mass spectrometric detection was performed in multiple reaction monitoring mode on 4000Q UFLC-MS/MS system with turbo ion spray source in a negative and positive switching ionization mode. The lower limits of quantification were 0.2-1.5 ng/ml for all the analytes. Both intra-day and inter-day precision and accuracy of analytes were well within acceptance criteria (±15%). The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 60.0%. The validated method has been successfully applied to comparing pharmacokinetic profiles of analytes in normal and AD rat plasma. The results indicated that no significant differences in pharmacokinetic parameters of GRe, GRg1 and TUM were observed between the two groups, while the absorption of POL and GRd in AD group were significantly higher than those in normal group; moreover, the GRb1 absorbed more rapidly in model group. The different characters of pharmacokinetics might be caused by pharmacological effects of the analytes.

Simultaneous determination of three alkaloids, four ginsenosides and limonin in the plasma of normal and headache rats after oral administration of Wu-Zhu-Yu decoction by a novel ultra fast liquid chromatography-tandem mass spectrometry method: application to a comparative pharmacokinetics and ethological study.

A novel, sensitive and reliable ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method has been developed and validated for simultaneous quantitation of eight main active ingredients (evodiamine, rutaecarpine, dehydroevodiamine, limonin, ginsenoside Rb1, Rd, Re and Rg1) in rat plasma after oral administration of Wu-Zhu-Yu (WZY) decoction, which is a celebrated and widely used Traditional Chinese Medicine formula for the treatment of headache. The analytes and internal standard (IS) were separated on a SHIM-PACK XR-ODS II column, and the detection was performed on a UFLC-MS/MS system with turbo ion spray source. The lower limits of quantification were 1.5, 0.5, 1.0, 2.0, 2.0, 1.0, 0.5 and 0.2 ng ml(-1) for evodiamine, rutaecarpine, dehydroevodiamine, limonin, gensenoside Rb1, Rd, Re and Rg1, respectively. Linearity, accuracy, precision and absolute recoveries of the eight analytes were all within satisfaction. The IS-normalized matrix factor was adopted for assessing the matrix effect and accompanied with a satisfactory result. The validated method has been successfully applied to compare pharmacokinetic profiles of the eight active ingredients in rat plasma between normal and headache rats after administration. Exact pharmaceutical effect of WZY decoction on headache was demonstrated by the ethological response of headache rats induced by nitric oxide donor after administration. The results indicated that the absorption of evodiamine, rutaecarpine, gensenoside Rb1, Re and Rg1 in headache group were significantly higher than those in normal group with similar concentration-time curves while no significant differences existed in limonin and ginsenoside Rd between the two groups.