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An assay based on protein precipitation and liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative analysis of lisinopril in human plasma. After the addition of enalaprilat as internal standard (IS), plasma samples were prepared by one-step protein precipitation using perchloric acid followed by an isocratic elution with 10 mm ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)-methanol (70:30, v/v) on a Phenomenex Luna 5 mu C(18) (2) column. Detection was performed on a triple-quadrupole mass spectrometer utilizing an electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 406 --> 246 for lisinopril and m/z 349 --> 206 for enalaprilat. Calibration curves of lisinopril in human plasma were linear (r = 0.9973-0.9998) over the concentration range 2-200 ng/mL with acceptable accuracy and precision. The limit of detection and lower limit of quantification in human plasma were 1 and 2 ng/mL, respectively. The validated LC-MS/MS method has been successfully applied to a preliminary pharmacokinetic study of lisinopril in Chinese healthy male volunteers.

Superparamagnetic iron oxide nanoparticle (SPION) holds great potential as a gene delivery system due to its unique properties, such as good biocompatibility and non-invasive targeting ability. In this study, we modified SPION with chitosan-graft-PEI (CHI-g-PEI) and PK11195, to fabricate a mitochondria-targeting gene carrier, PK-CP-SPION. PK-CP-SPION manifested prominent physicochemical properties for magnetic guided gene delivery, and it could effectively condense and protect DNA at proper weight ratios. The in vitro cytotoxicity of PK-CP-SPIONs was mild. Under an external magnetic field, the transfection efficiency of PK-CP-SPIONs was comparable to PEI 25 K with shorter transfection time. PK11195 facilitated the specific accumulation of PK-CP-SPIONs in mitochondria, leading to the leakage of cytochrome c, the dissipation of mitochondrial membrane potential and subsequently the activation of mitochondria apoptosis pathway. These results indicated that with further development, PK-CP-SPIONs could serve as a multifunctional nanoplatform for magnetic targeting gene delivery and mitochondria-targeting therapy, leading enhanced therapeutic effect towards tumor cells.

With a great difference in therapeutic effects of Mahuang (MH, the stems of Ephedra sinica) and Mahuanggen (MHG, the roots of Ephedra sinica), chemical differences between MH and MHG should be investigated. In the present study, gas chromatography-mass spectrometry (GC-MS)-based plant metabolomics was employed to compare volatile oil profiles of MH and MHG. The antioxidant activities of volatile oils from MH and MHG were also compared. 32 differential chemical markers were identified according to the variable importance in the projection (VIP) value of orthogonal partial least squares discriminant analysis (OPLS-DA) and P value of Mann-Whitney test. Among them, chemical markers of tetramethylpyrazine (TMP) and α-terpineol were quantified. Their contents were much higher in most MH samples compared with MHG. The antioxidant assay demonstrated that MH had significantly higher free radical-scavenging activity than MHG. Although MH and MHG derived from the same medicinal plant, there was much difference in their volatile oil profiles. MH samples had significantly higher content of two reported pharmacologically important chemical markers of TMP and α-terpineol, which may account for their different antioxidant activities.

Therapeutic effects of herbal medicines differ greatly due to the use of different anatomical parts or processing methods in traditional Chinese medicine, and Ephedra sinica (ES) is just a case in point. To better understand different traditional uses of the stems (known as Mahuang, MH) and roots (known as Mahuanggen, MHG) of ES, their therapeutic material basis should be investigated. In this study, ephedrine alkaloids were profiled simultaneously with primary metabolites using GC-MS based metabolomics. Ephedrine (E) has been reported to be the major bioactive constituent in MH for the treatment of asthma. The results showed that compared with MH, MHG contained much lower levels of five ephedrine alkaloids, which may well explain that MHG has not been used as an antiasthmatic. Additionally, these pharmacologically important ephedrine alkaloids exhibited strong positive correlation with five primary metabolites. In conclusion, this study facilitates better understanding of different traditional uses of MH and MHG.

A sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method for the simultaneous determination of spironolactone and its active metabolite canrenone in human plasma has been developed and validated. After the addition of estazolam as the internal standard (IS), plasma samples were extracted with methylene chloride : ethyl acetate mixture (20 : 80, v/v) and separated by high-performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of methanol-water (57 : 43, v/v). Analytes were determined in a single quadrupole mass spectrometer using an atmospheric pressure chemical ionization (APCI) source. LC-APCI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.25 for spironolactone and canrenone, m/z 295.05 for estazolam. The method was proved to be sensitive and specific by testing six different plasma batches. Calibration curves of spironolactone and canrenone were linear over the range 2-300 ng/ml. The within- and between-batch precisions (relative standard deviation (RSD)%) were lower than 10% and the accuracy ranged from 85 to 115%. The lower limit of quantification (LLOQ) was identifiable and reproducible at 2 ng/ml. The proposed method was successfully applied to study the pharmacokinetics of spironolactone and its major metabolite in healthy male Chinese volunteers.

This paper describes a novel method for the sensitive and selective determination of fudosteine in human plasma. The method involves a derivatization step with 9-fluorenylmethyl chloroformate (FMOC-Cl) in borate buffer and detection based on high-performance liquid chromatography-electrospray ionization mass spectrometry (LC/ESI/MS). After acetonitrile-induced protein precipitation of plasma samples, fudosteine was derivatized with FMOC-Cl, then extracted by ethyl acetate, evaporated, reconstituted and injected using an LC/ESI/MS instrument. Separation was achieved using an ODS column and isocratic elution. Excellent linearity was obtained for the entire calibration range from 0.05 to 20 microg/ml. Validation assays of the lower limit of quantification (LLOQ) as well as for the intra- and inter-batch precision and accuracy met the international acceptance criteria for bioanalytical method validation. Using the developed analytical method, fudosteine could be detected for the first time in human plasma with a low limit of detection (LLOD) of 0.03 microg/ml. The proposed method has been successfully applied to study the pharmacokinetics of fudosteine in healthy Chinese volunteers after single and multiple oral administration.

Ethnopharmacological relevance Danshen and Sanqi Prescription (Dan-Qi) is commonly used to treat cardiovascular diseases (CVD) in China. Since Danshen and Sanqi are reported to ameliorate lipid metabolism disorders at treatment of cardiovascular diseases. Meanwhile, it is reported that co-administration of Danshen and Sanqi exhibited significant pharmacokinetic herb–herb interactions. We reasoned that Danshen and Sanqi combination could be potentially function synergistically in treating diet induced insulin resistance. Materials and methods Using high calori food induced Drosophila and mice models, we assessed Danshen and Sanqi treatment for their anti-diabetic effects. Results The combination of Danshen and Sanqi (Dan-Qi) effectively improved fat and glucose metabolism of the high-sugar and high-fat diet fed fruit flies. More importantly, Dan-Qi significantly ameliorated hyperlipidemia and hyperglycemia phenotype caused in high-fat diet induced obesity (DIO) mouse model. The Dan-Qi treated DIO mice showed lower fasting insulin, triglycerides, total and low-density lipoprotein cholesterol (LDL-C) levels in plasma, much better than Danshen or Sanqi treated alone. It was shown that Dan-Qi prescription reduced fat accumulation in the liver with Sanqi playing the major role. Interestingly, it was not Danshen or Sanqi alone, but the combination markedly increased glycogen deposition in mice liver. Quantitative RT-PCR showed Dan-Qi increased liver glycogen synthesis gene like Glut-1, GK, and Glut-4, reduced fat and cholesterol anabolism genes such as SREBP-1c, ACC, ATP-CL, ACS. Meanwhilpose tissues and muscle tissues, the glucose and fat metabolism genes are changed accordingly to pro-catabolism status. Notably, endogenous plasma metabolites of Dan-Qi treated mice displayed much better overral rectifying effects than the Danshen or Sanqi alone. Conclusions Our data demonstrated that Danshen and Sanqi combination exerted significant anti-diabetic efficacy, and Dan-Qi prescription could be potentially considered as a therapeutic application in diabetes.

Correction for ‘Plant metabolomics driven chemical and biological comparison of the root bark of Dictamnus dasycarpus and Dictamnus angustifolius’ by Mengying Lv et al., RSC Adv., 2015, 5, 15700–15708.

A practical synthesis of α-amyrin (1), β-amyrin (2), and lupeol (3) was accomplished in total yields of 32, 42, and 40% starting from easily available ursolic acid (4), oleanolic acid (5), and betulin (6), respectively. Remarkably, these three natural pentacyclic triterpenes exhibited potential inhibitory activity against human oxidosqualene cyclase.

Crude rhubarb subjected to different processing procedures will produce different therapeutic effects that are possibly due to processing-induced variation in chemical composition. In this study, a chemometric approach based on ultra fast liquid chromatography with ion trap/time-of-flight mass spectrometry was established to systematically investigate the chemical variations of rhubarb induced by different processing methods. The approach was validated based on pooled quality-control samples from two perspectives: the individual properties of variables and the bulk properties of samples. Orthogonal partial least squares discriminant analysis was introduced to compare the differences between crude and processed rhubarb products. A total of 20 significantly different markers were screened out and unambiguously/tentatively characterized. This research proved that a chemometric method based on ultra fast liquid chromatography with ion trap/time-of-flight mass spectrometry can comprehensively analyze the chemical variation of herbal medicine and provide evidence for a deeper understanding of the pharmacological activities of processed rhubarb products.

A sensitive and simple liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the determination of corilagin in rat plasma has been developed. Samples were prepared with protein precipitation method and analyzed with a triple quadrupole tandem mass spectrometer. We employed negative electrospray ionization as the ionization source and the analytes were detected in multiple reaction monitoring mode. Separation was achieved on a C8 column eluted with mobile phase consisting of methanol-0.1% formic acid in a gradient mode at the flow rate of 0.3 mL/min. The total run time was 7.0 min.This method was proved to have good linearity in the concentration range of 2.5-1000.0 ng/mL. The lower limit of quantification of corilagin was 2.5 ng/mL. The intra- and inter-day relative standard deviationa across three validation runs for four concentration levels were both9.8%. The relative error was within ±6.0%. This assay offers advantages in terms of expediency and suitability for the analysis of corilagin in rat plasma. The practical utility of this new HPLC-MS/MS method was confirmed in pilot plasma concentration studies in rats following oral administration.

The anti-malarial drug artesunate possesses anti-inflammatory and anti-oxidative actions in experimental asthma, comparable to corticosteroid. We hypothesized that artesunate may modulate disease-relevant metabolic alterations in allergic asthma. To explore metabolic profile changes induced by artesunate in allergic airway inflammation, we analysed bronchoalveolar lavage fluid (BALF) and serum from naive and ovalbumin-induced asthma mice treated with artesunate, using both gas and liquid chromatography-mass spectrometry metabolomics. Pharmacokinetics analyses of serum and lung tissues revealed that artesunate is rapidly converted into the active metabolite dihydroartemisinin. Artesunate effectively suppressed BALF total and differential counts, and repressed BALF Th2 cytokines, IL-17, IL-12(p40), MCP-1 and G-CSF levels. Artesunate had no effects on both BALF and serum metabolome in naive mice. Artesunate promoted restoration of BALF sterols (cholesterol, cholic acid and cortol), phosphatidylcholines and carbohydrates (arabinose, mannose and galactose) and of serum 18-oxocortisol, galactose, glucose and glucouronic acid in asthma. Artesunate prevented OVA-induced increases in pro-inflammatory metabolites from arginine–proline metabolic pathway, particularly BALF levels of urea and alanine and serum levels of urea, proline, valine and homoserine. Multiple statistical correlation analyses revealed association between altered BALF and serum metabolites and inflammatory cytokines. Dexamethasone failed to reduce urea level and caused widespread changes in metabolites irrelevant to asthma development. Here we report the first metabolome profile of artesunate treatment in experimental asthma. Artesunate restored specific metabolic perturbations in airway inflammation, which correlated well with its anti-inflammatory actions. Our metabolomics findings further strengthen the therapeutic value of using artesunate to treat allergic asthma.

As an arsenical, realgar (As4S4) is known as a poison and paradoxically as a therapeutic agent. However, a complete understanding of the precise biochemical alterations accompanying the toxicity and therapy effects of realgar is lacking. Using a combined ultrafast liquid chromatography (UFLC) coupled with ion trap time-of-flight mass spectrometry (IT-TOF/MS) and (1)H NMR spectroscopy based metabolomics approach, we were able to delineate significantly altered metabolites in the urine samples of realgar-treated rats. The platform stability of the liquid chromatography LC/MS and NMR techniques was systematically investigated, and the data processing method was carefully optimized. Our results indicate significant perturbations in amino acid metabolism, citric acid cycle, choline metabolism, and porphyrin metabolism. Thirty-six metabolites were proposed as potential safety biomarkers related to disturbances caused by realgar, and glycine and serine are expected to serve as the central contacts in the metabolic pathways related to realgar-induced disturbance. The LC/MS and NMR based metabolomics approach established provided a systematic and holistic view of the biochemical effects of realgar on rats, and might be employed to investigate other drugs or xenobiotics in the future.

Differences in the contents of sixteen bioactive components (three tannins, five anthraquinones, six flavonoids and two neolignans) between Da-Cheng-Qi decoction (DCQD) and its three constitutional herbal medicines (Radix et Rhizoma Rhei, Cortex Magnoliae officinalis, and Fructus Aurantii Immaturus) were compared using validated HPLC/DAD methods. The results indicated that there existed some kinds of interactions between these constitutional natural medicines during the DCQD preparation procedure, which could either enhance or depress the extractive rates of bioactive components.

Da-Cheng-Qi decoction (DCQD) is a purgative compound prescription used in China and East Asia. In this paper, pharmacokinetic differences of six major active components (rhein, emodin, aloe-emodin, magnolol, naringenin and hesperetin) between DCQD and its three constitutional herbal medicines, i.e., Radix et Rhizoma Rhei, Cortex Magnoliae officinalis and Fructus Aurantii Immaturus were investigated in rats after oral administration. Plasma samples were analyzed for the quantification of the six active components using validated LC-MS/MS methods. Unpaired Student's t-test was used for statistical comparison. Significant differences (p0.05) in the main pharmacokinetic parameters for rhein, emodin, aloeemodin, magnolol, naringenin and hesperetin were found between DCQD and the decoction of its constitutional single herbal medicines, which demonstrated the presence of drug-drug interactions between these constitutional raw materials of DCQD occurring either in the procedure of decoction or during ADME process.

Da-Cheng-Qi decoction (DCQD) is a purgative prescription used in China and East Asia. To profile the constituents of this complex traditional Chinese medicine (TCM), a high-performance liquid chromatographic, electrospray ionization, tandem mass spectrometric (HPLC-ESI/MS/MS) analytical method was developed. After separation on a reversed-phase C18 analytical column using gradient elution, samples were analyzed by ESI-MS/MS in negative mode. As a result, a total of 37 compounds were detected, of which two tannins, three anthraquinones, two sennosides, five flavonoids and two lignans were unambiguously identified by comparison with standard compounds, and sixteen compounds were either tentatively identified or deduced according to their MS/MS data. The fragmentation pathways of many of the observed compounds, such as the tannins and lignans are reported for the first time. In addition, the identity of each peak in DCQD was explored by comparison with those of its three constituent herbs. The results indicated that tannins, anthraquinones and sennosides in DCQD originated from Radix et Rhizoma Rhei, flavonoids from Fructus Aurantii Immaturus, and lignans from Cortex Magnoliae officinalis. The present study provides an example of chemical constitution profiling in complex TCM systems using LC/MS/MS.

The integration of liquid chromatography-mass spectrometry (LC-MS) with derivatization is a relatively new and unique strategy that could add value and could enhance the capabilities of LC-MS-based technologies. The derivatization process could be carried out in various analytical steps, for example, sampling, storage, sample preparation, HPLC separation, and MS detection. This review presents an overview of derivatization-based LC-MS strategy over the past 10 years and covers both the general principles and applications in the fields of pharmaceutical and biomedical analysis, biomarker and metabolomic research, environmental analysis, and food-safety evaluation. The underlying mechanisms and theories for derivative reagent selection are summarized and highlighted to guide future studies.

High-performance liquid chromatography with diode-array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) was used for separation and identification of metabolites in rat urine, bile and plasma after oral administration of rhubarb decoction. Based on the proposed strategy, 91 of the 113 potential metabolites were tentatively identified or characterized. Besides anthraquinones metabolites, gallic acid, (-)-epicatechin and (+)-catechin metabolites were also detected and characterized in these biological samples. Our results indicated that glucuronidation and sulfation were the main metabolic pathways of anthraquinones, while methylation, glucuronidation and sulfation were the main metabolic pathways of gallic acid, (-)-epicatechin and (+)-catechin. Phase I reactions (e.g., hydroxylation and reduction) played a relatively minor role compared to phase II reactions in metabolism of phenolic compounds of rhubarb decoction. The identification and structure elucidation of these metabolites provided essential data for further pharmacological and clinical studies of rhubarb and related preparations. Moreover, the results of the present investigations clearly indicated the relevance and usefulness of the combination of chromatographic, spectrophotometric, and mass-spectrometric analysis to detect and identify metabolites.

The aim is to develop a simple and specific method for the extraction and chemical fingerprinting of Andrographis paniculata (Burm. f.) Nees and to apply the method to this drug from different regions. High-performance liquid chromatographic (HPLC) with gradient elution is used for developing the fingerprints, and liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) technique is employed to identify the component of the fingerprints. Nine peaks are selected as common peaks, and six compounds are elucidated by MS data. Twenty-three samples of A. paniculata from different regions of China are collected and detected by HPLC fingerprinting. Comparisons of the chromatograms show that there are obvious differences in the content of each component contained between the habitat samples in China. The results of hierarchical cluster analysis show that these samples can be clustered reasonably into three groups, and the growth of A. paniculata and their internal quality are related to their habitat. The HPLC fingerprint developed allows simple identification of A. paniculata from many natural drugs.

In this work a systematic strategy integrating liquid chromatography/tandem mass spectrometry (LC/MS/MS) and online databases was developed to identify phosphocholines (PC) and lysophosphatidylcholines (LPC) in human red blood cells (RBCs). First of all, the neutral loss scan of 59 and the precursor ion scan of m/z 184 were performed to find out the possible lipids with phosphocholine head-group structure in RBCs. The acquired [M+H](+) and [M+Na](+) adduct ions were then identified online using the Human Metabolome Database (HMDB) and the LIPID MAPS, which were then further confirmed by their MS/MS fragmentation. Based on the comparison of chemical structures of the detected PC and LPC with their corresponding MS/MS fragmentation pathways, several new diagnostic fragments or fragmentation pathway were found. These include, (1) the neutral losses of 183 could be used as a diagnostic fragmentation to discriminate PC and LPC; (2) product ions at m/z 104 could be used to distinguish LPC and their sn-2 isomers; (3) fragment ions at m/z 184 are characteristic fragmentation that could be used for discrimination of sodiated ions [M+Na](+) and protonated ions [M+H](+), especially for co-eluted PC or LPC with a molecular weight difference of 22. The structures of the above-mentioned fragment ions were confirmed by quadrupole time-of-flight (Q-TOF) MS. Furthermore, a PC and LPC focused LC/MS semi-quantification approach was also developed and validated. This approach could be useful for future lipidomic study.

Background Da Chengqi Tang (DCT) is a common purgative formula in Chinese medicine. Flavanones are its major active compounds derived from Fructus Aurantii Immaturus. The present study developed an LC-MS/MS method to characterize two pairs of flavanone 7-glycoside isomers, i.e., hesperidin versus neohesperidin and naringin versus isonaringin. Methods After solid phase purification, components in sample were separated on a Agilent zorbax SB-C18 (5 μm, 250 mm × 4.6 mm) analytical column. ESI-MS and quasi-MSn were performed in negative ion mode to obtain structural data of these two pairs of flavanone 7-glycoside isomers. Moreover, UV absorption was measured. Results There was no intra-pairs difference in the UV-Vis and MS/MS spectra of the two pairs of 7-glycoside isomers, whereas the mass spectrometry fragmentation pathways between pairs were different. Conclusion The present study developed a LC-MS/MS method to explore the inter- and intra-pair difference of two pairs of flavanone 7-glycoside isomers.

As the unique quality control standard of Tabellae Glucosidorum Tripterygii Totorum, triptolide (CAS 38748-32-2) has a narrow therapeutic window. A significant side-effect of triptolide is its male reproductive toxicity the mechanism of which is still unknown. Therefore, in the present study the male reproductive toxicity and toxicokinetics of triptolide were investigated. Male Sprague-Dawley (SD) rats were treated with triptolide by oral administration (gastric infusion; 0, 100, 200, 400 microg/kg) once daily for 8 weeks. At the end of the treatment, the concentrations of triptolide in blood and testis samples were analyzed with liquid chromatography/mass spectrometry (LC/MS) to obtain the toxicokinetic parameters. Triptolide showed a non-linear kinetics profile and was rapidly absorbed but relatively slowly eliminated in the rats. Specifically, an accumulation of triptolide was seen in the testis. In the hematological study, mean corpuscular hemoglobin concentration (MCHC) had a marginal decrease in all triptolide treated groups. Alkaline phosphatase (ALP) in serum increased significantly only in the 400 microg/kg group during clinical chemistry assays although no histopathological change was found in the hematopoietic system or liver. In the male reproductive toxicity studies, the testis and epididymis weights of all triptolide treatment groups decreased significantly. The cauda epididymis sperm content and motility even decreased to zero. Evident changes were observed in the seminiferous tubules and the epididymides of triptolide treated rats (e.g., intraepithelial vacuoles of varying sizes; increased germ cells de generation, exfoliation and tubular atrophy). These findings provide valuable information to estimate the reproductive risk of triptolide in humans.

A sensitive and specific liquid chromatography-electrospray ionization mass spectrometry method is developed and validated for the identification and quantitation of azithromycin in human plasma. After the addition of the internal standard and 1.0M sodium hydroxide solution, plasma samples are extracted with a methylene chloride-ethyl acetate mixture (20:80, v/v). The organic layer is evaporated under a stream of nitrogen at 40 degrees C. The residue is reconstituted with 200 microL of the mobile phase. The compounds are separated on a prepacked Shimadzu Shim-pack VP-ODS C18 (5 microm, 150 mm x 2.0 mm) column using a mixture of acetonitrile-water (65:35) (0.5% triethylamine, pH was adjusted to 6.2 with acetic acid) as the mobile phase. Detection is performed on a single quadrupole mass spectrometer by selected ion monitoring mode via electrospray ionization source. The method is fully validated and linear calibration curves are obtained in the concentration ranges from 5 to 2000 ng/mL. The intra- and inter-batch relative standard deviations at four different concentration levels are all < 10%. The limit of detection and quantitation are 2 ng/mL and 5 ng/mL, respectively. The proposed method enables the unambiguous identification and quantitation of azithromycin for pharmacokinetic, bioavailability, or bioequivalence studies.

ZLR-8 is a nitric oxide releasing derivative of diclofenac for the treatment of inflammation. In this paper, a sensitive and reliable high-performance liquid chromatography method for simultaneous determination of ZLR-8 and its active metabolite diclofenac in the plasma of beagle dogs has been developed and validated. After the addition of ketoprofen as the internal standard (IS), plasma samples were extracted with n-hexane-isopropanol (95:5, v/v) mixture solution and separated by HPLC on a reversed-phase C(18) column with a mobile phase of gradient procedure. Analytes were determined by the UV detector which was set at 280 nm. The method was proved to be sensitive and specific by testing six different plasma batches. Calibration curves of ZLR-8 and diclofenac were linear over the range 0.05-4.0 microg/mL. The within- and between-batch precisions (RSD%) were lower than 10% and accuracy ranged from 85 to 115%. The lower limit of quantification was identifiable and reproducible at 0.05 microg/mL. The proposed method has been readily implemented in preclinical pharmacokinetics studies of ZLR-8 and its active metabolite diclofeance. Representative plasma concentration vs time profiles resulting from administration of ZLR-8 to beagle dogs are presented in this communication.

The pharmacokinetics of fudosteine in healthy Chinese volunteers was investigated for the first time after single- and multiple-dose administration. Five male and five female volunteers were enrolled in this study. Each subject received 400 mg fudosteine capsules (the therapeutic dose) on day 1 after overnight fasting for the single-dose study and three times daily oral administration (400 mg) for 5 consecutive days until the sixth morning for the multiple-dose study. Serial blood samples were collected at specified time intervals up to 16 hours following the first and last doses of fudosteine. Plasma harvested from the blood was separated and analyzed for fudosteine levels by a validated high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI/MS) method employing percolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). Noncompartmental analysis was used for the calculation of the total area under the plasma concentration-time curve (AUC) from time zero to time infinity and the terminal half-life (t1/2) of fudosteine. The pharmacokinetic parameters for single- and multiple-dose administration were estimated as follows: Cmax amounted to 10.13+/-4.39 microg/mL and 11.75+/-6.51 microg/mL, tmax to 0.69+/-0.36 h and 0.53+/-0.12 h and t1/2 to 2.33+/-0.63 h and 2.40+/-0.37 h, respectively. No significant differences were found between single- and multiple-dose oral administration, although gender differences were observed.

A selective and sensitive high performance liquid chromatography-electrospray ionisation-mass spectrometry method has been developed for the determination of balofloxacin (BLFX) in human plasma. The sample preparation was a liquid-liquid extraction, and chromatographic separation was achieved with an Agilent ZORBAX 300SB C18 2.1 mm x 150 mm column using a mobile phase comprised of methanol-water (10 mM CH(3)COONH(4), pH 3.0)=40:60 (v/v). Standard curves were linear (r=0.9992) over the concentration range of 0.03-3 microg/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation (R.S.D.). The limit of detection (LOD) was 0.02 microg/ml. The validated HPLC-electrospray ionization (ESI)-MS method has been used successfully to study balofloxacin pharmacokinetics in healthy volunteers.

A rapid, sensitive and specific high-performance liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) method was developed and validated for the first time to determine the concentration of lafutidine in human plasma. After the addition of diazepam (the internal standard, IS) and 1 M sodium hydroxide solution to 0.5-ml plasma sample, lafutidine was extracted from plasma with n-hexane : isopropanol (95 : 5, v/v). The organic layer was evaporated and the residue was redissolved in 200-microl mobile phase. The analyte was chromatographically separated on a prepacked Shimadzu Shim-pack VP-ODS C(18) column (250 x 2.0 mm i.d.) using a mixture of methanol-water (20 mM CH(3)COONH(4)) = 80 : 20 (v/v) as mobile phase. Detection was performed on a single quadrupole mass spectrometer using an electrospray ionization interface and the selected-ion monitoring (SIM) mode. The method showed excellent linearity (r = 0.9993) over the concentration range of 5-400 ng/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation. The limit of detection was 1 ng/ml. The validated LC/ESI-MS method has been successfully applied to the bioequivalence study of lafutidine in 24 healthy male Chinese volunteers.

A sensitive and specific liquid chromatography–atmospheric pressure chemical ionisation-mass spectrometry (LC–APCI-MS) method has been developed and validated for the identification and quantification of zaleplon in human plasma using estazolam as an internal standard (IS). After the addition of estazolam and 2.0 M sodium hydroxide solution, plasma samples were extracted with ethyl acetate and then the organic layer was evaporated to dryness. The reconstituted solution of the residue was injected onto a prepacked Shim-pack VP-ODS C 18 (250 mm × 2.0 mm i.d.) column and chromatographed with a mobile phase comprised of methanol:water (70:30) at a flow-rate of 0.2 ml/min. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via atmospheric pressure chemical ionization (APCI) source. The mean standard curve was linear ( r = 0.9991) over the concentration range of 0.2–100 ng/ml and had good back-calculated accuracy and precision. The intra-day and inter-day precisions were within 10% relative standard deviation and accuracy ranged from 85% to 115%. The limit of detection was 0.1 ng/ml. The validated LC–APCI-MS method has been used successfully to study zaleplon pharmacokinetic, bioavailability and bioequivalence in 18 adult volunteers.

A selective and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for the determination of tolterodine tartrate in human plasma. With oxybutynin as internal standard, tolterodine tartrate was extracted from plasma with n-hexane: isopropanol (95:5, v/v). The organic layer was evaporated and the residue was redissolved in mobile phase comprised of acetonitrile-water (10 mM CH3COONH4, pH 3.0)=50:50 (v/v). An aliquot of 10 microl was chromatographically analyzed on a prepacked Shimadzu Shim-pack VP-ODS C18 column (150 mmx2.0 mm I.D.) by means of selected-ion monitoring (SIM) mode mass spectrometry. Standard curves were linear (r=0.9993) over the concentration range of 0.1-30.0 ng/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation. The limit of detection (LOD) was 0.05 ng/ml. The validated LC-ESI-MS method has been used successfully to study tolterodine tartrate pharmacokinetic, bioavailability and bioequivalence in 20 healthy male volunteers.

A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of tulobuterol in rabbits' plasma. After the addition of clenbuterol-HCl, the internal standard (IS) and 1.0 M sodium hydroxide solution, plasma samples were extracted using a solvent mixture comprised of 5% isopropanol in n-hexane. The compounds were separated on a prepacked Lichrospher CN (5 microm, 150 mm x 2.0 mm) column using a mixture of methanol-water (10 mM CH3COONH4, pH 4.0) as mobile phase. A Shimadzu LCMS-2010A mass spectrometer connected to a Shimadzu high performance liquid chromatograph (HPLC) was used to develop and validate the method. The method has shown to be sensitive and specific by testing six different blank plasma batches. Linearity was established for the range of concentrations 0.50-40.0 ng/mL with a coefficient of determination (r) of 0.9998. The intra-day precision was better than 15%. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.50 ng/mL. The proposed method enables the unambiguous identification and quantification of tulobuterol for pharmacokinetic, bioavailability or bioequivalence studies.

A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of zolmitriptan in human plasma. After the addition of the internal standard (IS) and 1.0 M sodium hydroxide solution, plasma samples were extracted with methylene chloride:ethyl acetate mixture (20:80, v/v). The organic layer was evaporated under a stream of nitrogen at 40 degrees C. The residue was reconstituted with 100 microl mobile phase. The compounds were separated on a prepacked Lichrospher CN (5 microm, 150 mm x 2.0 mm) column using a mixture of methanol:water (10 mM NH(4)AC, pH 4.0) = 78:22 as mobile phase. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 0.30-16.0 ng/ml with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision (R.S.D.%) were lower than 15% and accuracy ranged from 85 to 115%. The lower limit of quantification was identifiable and reproducible at 0.30 ng/ml. The proposed method enables the unambiguous identification and quantification of zolmitriptan for pharmacokinetic, bioavailability or bioequivalence studies.

Background: Dengue virus (DENV) is the most widespread arbovirus with an estimated 100 million infections occurring every year. Endemic in the tropical and subtropical areas of the world, dengue fever/dengue hemorrhagic fever (DF/DHF) is emerging as a major public health concern. The complex array of concurrent host physiologic changes has hampered a complete understanding of underlying molecular mechanisms of dengue pathogenesis. Methodology/Principle Findings: Systems level characterization of serum metabolome and lipidome of adult DF patients at early febrile, defervescence, and convalescent stages of DENV infection was performed using liquid chromatography- and gas chromatography-mass spectrometry. The tractability of following metabolite and lipid changes in a relatively large sample size (n = 44) across three prominent infection stages allowed the identification of critical physiologic changes that coincided with the different stages. Sixty differential metabolites were identified in our metabolomics analysis and the main metabolite classes were free fatty acids, acylcarnitines, phospholipids, and amino acids. Major perturbed metabolic pathways included fatty acid biosynthesis and β-oxidation, phospholipid catabolism, steroid hormone pathway, etc., suggesting the multifactorial nature of human host responses. Analysis of phospholipids and sphingolipids verified the temporal trends and revealed association with lymphocytes and platelets numbers. These metabolites were significantly perturbed during the early stages, and normalized to control levels at convalescent stage, suggesting their potential utility as prognostic markers. Conclusions/Significance: DENV infection causes temporally distinct serum metabolome and lipidome changes, and many of the differential metabolites are involved in acute inflammatory responses. Our global analyses revealed early anti-inflammatory responses working in concert to modulate early pro-inflammatory processes, thus preventing the host from development of pathologies by excessive or prolonged inflammation. This study is the first example of how an omic- approach can divulge the extensive, concurrent, and dynamic host responses elicited by DENV and offers plausible physiological insights to why DF is self limiting., Singapore-MIT Alliance for Research and Technology

Zebrafish embryogenesis is a rapid process driven by a myriad of gene products and small molecules. As previous studies have detailed the relevant transcriptional and proteomics changes, here we assess the metabolomic changes that occur at different stages of embryogenesis (4, 8, 12, 24 and 48 hours post fertilization). Metabolite levels were detected using GC-MS and LC-MS, following which multivariate analysis (OPLS-DA) was applied to identify metabolites that were differentially regulated throughout embryogenesis. From the two robust OPLS-DA models that were generated (Q(2)(cum) = 0.940 and Q(2)(cum) = 0.894), a total of 60 detected metabolites (20 from GC-MS, 40 from LC-MS) were identified and found to be important in discriminating between developmental stages. Hierarchical clustering analysis was applied to the dataset and metabolite classes such as amino acids and lipids were shown to be differentially regulated. Biologically relevant transcriptomic and proteomic data were associated with metabolites to provide a more holistic systems perspective of embryogenesis. In summary, the metabolic profiles of different developmental stages highlight the dynamic changes occurring during embryogenesis. These data could serve as a basis for future toxicological or developmental studies.

Oxidative stress is an important clinical feature of asthma, resulting in oxidative damage to lung proteins, DNA and lipids. Our previous study has shown that artesunate, an anti-malarial drug, has potent anti-inflammatory effects in a murine asthma model. As oxidative stress plays a critical role in the pathogenesis of asthma, we further investigated the effects of artesunate on oxidative lung damage in this asthma model. BALB/c mice sensitized and challenged with ovalbumin, developed airway inflammation and oxidative lung damage with increased levels of nitrotyrosine (protein), 8-isoprostane (lipid) and 8-hydroxy-2-deoxy-guanosine (DNA). Superoxide dismutase (SOD) expression and activity were increased, while catalase expression and activity were suppressed in asthmatic mice. Treatment with artesunate showed comparable anti-inflammatory effects to dexamethasone, a potent corticosteriod, significantly suppressing airway inflammation and oxidative lung damage. Mice treated with artesunate had reduced levels of nitrotyrosine, 8-isoprostane and 8-hydroxy-2-deoxy-guanosine. SOD expression and activity were returned to basal levels, while catalase gene expression and activity were promoted with artesunate treatment. Gluthathione peroxidase expression and activity were unaltered in all treatment groups. Our findings indicate that artesunate has potent anti-inflammatory effects, comparable to dexamethasone, by ameliorating oxidative lung damage in experimental allergic airways.

Differences in the contents of sixteen bioactive components (three tannins, five anthraquinones, six flavonoids and two neolignans) between Da-Cheng-Qi decoction (DCQD) and its three constitutional herbal medicines (Radix et Rhizoma Rhei, Cortex Magnoliae officinalis, and Fructus Aurantii Immaturus) were compared using validated HPLC/DAD methods. The results indicated that there existed some kinds of interactions between these constitutional natural medicines during the DCQD preparation procedure, which could either enhance or depress the extractive rates of bioactive components.

Da-Cheng-Qi decoction (DCQD) is a purgative prescription used in China and East Asia. To profile the constituents of this complex traditional Chinese medicine (TCM), a high-performance liquid chromatographic, electrospray ionization, tandem mass spectrometric (HPLC-ESI/MS/MS) analytical method was developed. After separation on a reversed-phase C18 analytical column using gradient elution, samples were analyzed by ESI-MS/MS in negative mode. As a result, a total of 37 compounds were detected, of which two tannins, three anthraquinones, two sennosides, five flavonoids and two lignans were unambiguously identified by comparison with standard compounds, and sixteen compounds were either tentatively identified or deduced according to their MS/MS data. The fragmentation pathways of many of the observed compounds, such as the tannins and lignans are reported for the first time. In addition, the identity of each peak in DCQD was explored by comparison with those of its three constituent herbs. The results indicated that tannins, anthraquinones and sennosides in DCQD originated from Radix et Rhizoma Rhei, flavonoids from Fructus Aurantii Immaturus, and lignans from Cortex Magnoliae officinalis. The present study provides an example of chemical constitution profiling in complex TCM systems using LC/MS/MS.