High-performance Liquid Chromatography-electrospray Ionization-tandem Mass Research Articles

Incidence of Pyrrolizidine Alkaloids in Herbal Medicines from German Retail Markets: Risk Assessments and Implications to Consumers.

The occurrence of potentially toxic pyrrolizidine alkaloids (PAs) in herbal medicines (HMs) is currently intensely being discussed in Europe. Pyrrolizidine alkaloids, particularly the 1,2-unsaturated PAs, are undesired compounds in HMs due to their potential hepatotoxic and carcinogenic properties. In this study, 98 widely patronized HMs from six popular German retail supermarkets/drugstores, as well as from pharmacies, were analyzed by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the presence of PAs. The results showed that about 63% of the HMs were PA positive, whereas the average PA concentration of the samples was 201μg/kg, the highest concentration of PAs (3270μg/kg) was attributed to a product that was purchased from the pharmacy and contained Hypericum perforatum L. (St. John's Wort) as an active ingredient. In addition, H. perforatum-containing products were frequently contaminated with PAs from Echium spp., while both Cynara cardunculus L. products and fixed-combination products of Gentiana lutea L., Rumex acetosa L., Verbena officinalis L., Sambucus nigra L., and Primula veris L. products were commonly contaminated with PAs of Senecio spp. The study showed that H. perforatum, C. cardunculus, Urtica dioica L., and fixed-combination products were frequently contaminated with PA levels above the recommended values of both the German and European Medicines Agencies. Copyright © 2017 John Wiley & Sons, Ltd.

Ascaroside Profiling of Caenorhabditis elegans Using Gas Chromatography–Electron Ionization Mass Spectrometry

Nematodes such as the model organism Caenorhabditis elegans produce various homologous series of l-ascarylose-derived glycolipids called ascarosides, which include several highly potent signals in intra and interspecies communication as well as cross-kingdom interactions. Given their low concentrations and large number of structurally similar components, mass spectrometric screens based on high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) are commonly employed for ascaroside detection and quantification. Here, we describe a complementary gas chromatography-electron ionization mass spectrometry (GC-EIMS) screen that utilizes an ascarylose-derived K1-fragment ion signal at m/z 130.1 [C6H14OSi]+● to highlight known as well as yet unidentified ascaroside components in TMS-derivatized crude nematode exometabolome extracts. GC-EIMS-based ascaroside profiling of wild-type and mutant C. elegans facilitates the analysis of all basic ascarosides using the same ionization technique while providing excellent resolution for the complete homologous series with side chains ranging from 3 to 33 carbons. Combined screening for m/z 130.1 along with side chain-specific J1 [M - 173]+ and J2 [M - 291]+ fragment ions, as well as additional characteristic marker ions from α-cleavage, enables convenient structure assignment of ca. 200 components from wild-type and peroxisomal β-oxidation mutants including (ω - 1)-linked acyl, enoyl, β-hydroxyacyl, and 2-ketoalkyl ascarosides along with their (ω)-linked or α-methyl isomers and ethanolamide derivatives, as well as 2-hydroxyalkyl ascarosides. Given the widespread availability of GC-MS and its increasing popularity in metabolomics, this method will promote the identification of ascarosides in C. elegans and other nematodes.

Gene expression of glycyrrhizin acid and accumulation of endogenous signaling molecule in Glycyrrhiza uralensis Fisch adventitious roots after Saccharomyces cerevisiae and Meyerozyma guilliermondii applications.

This study reports the best culture conditions for roots growth and accumulation of active components by optimizing the parameters. Glycyrrhiza uralensis adventitious roots metabolites were significantly increased after adding Saccharomyces cerevisiae and Meyerozyma guilliermondii. The highest contents of polysaccharide, glycyrrhizic acid, glycyrrhetinic acid, and total flavonoids were obtained in M. guilliermondii group; the content of glycyrrhizic acid was 5.3-fold higher than the control. In control and treatment groups, 12 compounds were identified by high-performance liquidchromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS), among which some new compounds have been detected in elicitor groups including 5,7-dihydroxyflavanone, glycyrrhisoflavanone, licorice saponin J2, uralsaponin B, (3R)-vestitol, and uralenol. Meyerozyma guilliermondii significantly upregulated the expression of the genes such as 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl diphosphate synthase, geranyl diphosphate synthase, squalene synthase, squalene epoxidase, β-amyrin synthase, and CYP88D6 and CYP72A154. Meanwhile, it increased the biosynthesis of signaling molecules (nitric oxide, salicylic acid, and jasmonic acid) in defense mechanism.

Flavone polyphenols dominate in Thymus schimperi Ronniger : LC–ESI–MS/MS characterization and study of anti-proliferative effects of plant extract on AGS and HepG2 cancer cells

Thymus schimperi is a highly localized and a rare plant endemic to Ethiopia. An optimized and validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method was applied to characterize 23 polyphenolic compounds found in ethyl acetate extracts of the plant. From those, flavones dominated and luteolin was the major component contributing 21.83% of the total composition (or 46.05±0.59g/kg of fresh sample weight). Validation data showed a determination coefficient (R2)≥0.997. Limits of detection (LOD) and quantification (LOQ) were 0.03-0.97 and 0.11-3.23mg/L, while recovery values spiked at 5 and 50mg/L were between 70.89-115.39 and 67.65-120.19%, respectively. Except for caffeic acid and epicatechin gallate, the relative standard deviations (%RSDs) were far below 15%, showing acceptable precision values. The plant extracts inhibited cell proliferation and induced cell death in human gastric adenocarcinoma (AGS) and liver hepatocellular carcinoma (HepG2) cancer cells. This is the first report of polyphenolic components from T. schimperi being characterized using HPLC-ESI-MS/MS. Being components of many edible vegetables, fruits, and spices, the identified polyphenols suggest that T. schimperi could be a potential food with promising health benefits.

Analysis of a broad range of perfluoroalkyl acids in accipiter feathers: method optimization and their occurrence in Nam Co Basin, Tibetan Plateau.

Perfluoroalkyl acids (PFAAs) are ubiquitous in the environment. They are prone to accumulate in organisms and have raised public attention in recent decades. Feather samples have been successfully applied as nondestructive indicators for several contaminants. However, a sophisticated analytical method for determining PFAAs in feathers is still lacking. In the present study, a series of conditions, such as the use of the solid-phase extraction cartridge type and extraction/digestion methods, were optimized for the analysis of 13 PFAAs in feathers. According to the spiked recoveries, a weak-anion exchange cartridge was chosen and the methanol was selected as the extraction solvent. In the present study, an optimized pretreatment procedure combined with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometric (HPLC-ESI-MS/MS) method was established for the determination of PFAAs in feathers. The recoveries and method detection limits of the PFAAs ranged from 71 to 120% and 0.16 to 0.54ng/g, respectively. Finally, 13 PFAAs in four accipiter feather samples from Nam Co Basin, Tibetan Plateau, were analyzed, indicating that PFOS was the predominant PFAA in accipiter feathers, with an average of 4.67ng/g, followed by the short-chain PFAAs, PFBS and PFBA, with averages of 1.91 and 1.39ng/g, respectively. These results partly indicated the current situation of PFAA pollution in the Nam Co Basin, especially the existence of short-chain PFAAs in this region.

Sensitive determination of thiols in wine samples by a stable isotope-coded derivatization reagent d0/d4-acridone-10-ethyl-N-maleimide coupled with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis

As the key aroma compounds, varietal thiols are the crucial odorants responsible for the flavor of wines. Quantitative analysis of thiols can provide crucial information for the aroma profiles of different wine styles. In this study, a rapid and sensitive method for the simultaneous determination of six thiols in wine using d0/d4-acridone-10-ethyl-N-maleimide (d0/d4-AENM) as stable isotope-coded derivatization reagent (SICD) by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed. Quantification of thiols was performed by using d4-AENM labeled thiols as the internal standards (IS), followed by stable isotope dilution HPLC-ESI-MS/MS analysis. The AENM derivatization combined with multiple reactions monitoring (MRM) not only allowed trace analysis of thiols due to the extremely high sensitivity, but also efficiently corrected the matrix effects during HPLC-MS/MS and the fluctuation in MS/MS signal intensity due to instrument. The obtained internal standard calibration curves for six thiols were linear over the range of 25–10,000pmol/L (R2≥0.9961). Detection limits (LODs) for most of analytes were below 6.3pmol/L. The proposed method was successfully applied for the simultaneous determination of six kinds of thiols in wine samples with precisions ≤3.5% and recoveries ≥78.1%. In conclusion, the developed method is expected to be a promising tool for detection of trace thiols in wine and also in other complex matrix.

Characterization of glycerophospholipid molecular species in six species of edible clams by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

The molecular species of glycerophosphocholine (GPCho), glycerophosphoethanolamine (GPEtn), glycerophosphoserine (GPSer), lysoglycerophosphocholine (LGPCho) and lysoglycerophosphoethanolamine (LGPEtn) from six species of edible clams were characterized by using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. At least 435, 453, 468, 443, 427 and 444 glycerophospholipid (GP) molecular species were characterized, respectively, from Cyclina sinensis, Mactra chinensis Philippi, Mactra veneriformis Reeve, Meretrix meretrix, Ruditapes philippinarum and Saxidomus purpurata. Most of the predominant GP molecular species in clam contained polyunsaturated fatty acids (PUFA), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), indicating that clam is a potential resource of GP enriched PUFA. According to the amount of the major molecular species containing EPA and DHA, Cyclina sinensis was the best fit species for GPCho, Mactra veneriformis Reeve was the best fit species for GPEtn, Mactra chinensis Philippi was the best fit species for GPSer and LGPEtn, and Saxidomus purpurata was the best fit species for LGPCho.

Assessment of genetic fidelity and composition: Mixed elicitors enhance triterpenoid and flavonoid biosynthesis of Glycyrrhiza uralensis Fisch. tissue cultures.

Glycyrrhiza uralensis has acquired significant importance due to its medicinal properties and health function. In this study, the quality of G. uralensis adventitious roots was evaluated in terms of genetic stability, active compounds, and anti-inflammatory activity. Monomorphic banding pattern obtained from the mother plant and tissue cultures of G. uralensis with randomly amplified polymorphic DNA markers confirmed the genetic stability of adventitious roots. Neoliquiritin (neoisoliquiritin), ononin, liquiritin, and glycyrrhizic acid were identified from G. uralensis adventitious roots on the basis of high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. This study also revealed that adventitious roots possessed a better anti-inflammatory effect than native roots. To increase the contents of G. uralensis active components, elicitors were used in the adventitious roots culture. The combination of methyl jasmonate and phenylalanine synergistically stimulated the accumulation of glycyrrhetinic acid (0.22 mg/g) and total flavonoid (5.43 mg/g) compared with single treatment. In conclusion, G. uralensis adventitious roots can be an exploitable system for the production of licorice.

Identification of glycerophospholipid molecular species of mussel (Mytilus edulis) lipids by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

This study was carried out to identify the glycerophosphocholine (GPCho), glycerophosphoethanolamine (GPEtn) and glycerophosphoserine (GPSer) compositions in lipids extracted from mussels using Folch, Bligh-Dyer and methyl-tert-butyl ether (MTBE) methods by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). The molecular species of GPCho, GPEtn and GPSer were characterized according to the MS and MS/MS information. A semi-quantitative method using internal standard was established to compare the difference in glycerophospholipids (GP) between samples recovered with different methods. At least 212, 230 and 206 GP species were identified, respectively, from lipids recovered by Folch, Bligh-Dyer and MTBE methods. Most of the abundant GP species in mussels contained EPA and DHA. Some GP species with low content were not present in lipids recovered by the Folch and MTBE methods when compared with that recovered by the Bligh-Dyer method. However, for most GP species in lipids recovered by different methods, no quantitative differences existed.

Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases.

Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and their relative substrate selectivities.

Jasmonic acid and methyl dihydrojasmonate enhance saponin biosynthesis as well as expression of functional genes in adventitious roots of Panax notoginseng F.H. Chen.

Panax notoginseng, an important herbal medicine, has wide uses for its bioactive compounds and health function. In this work, we compared the content of saponin in cultivation and adventitious root. The total content of saponins in adventitious root (8.48mg⋅g-1 ) was found lower than in the native one (3-year-old) (34.34mg⋅g-1 ). To enhance the content of bioactive compounds, we applied elicitors jasmonic acid (JA) and methyl dihydrojasmonate (MDJ) to the adventitious root culture. It was observed that the highest total content of saponins (71.94mg⋅g-1 ) was achieved after treatment with 5mg⋅L-1 JA, which was 2.09-fold higher than native roots and 8.45-fold higher than the control group. The findings from high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis showed that six new compounds were present after the treatment with the elicitors. Furthermore, we found that JA and MDJ significantly upregulated the expression of the geranyl diphosphate synthase, farnesyl diphosphate synthase, squalene synthase, squalene epoxidase, dammarenediol synthase, and CYP716A47 and CYP716A53v2 (CYP450 enzyme) genes; downregulated the expression of the cycloartenol synthase gene; and increased superoxide dismutase and peroxidase activities.

UV photolysis of diclofenac in water; kinetics, degradation pathway and environmental aspects.

In this study, the photolysis behavior of commonly used anti-inflammatory drug diclofenac (DCF) was investigated using UV-C and UV-A irradiation. In that purpose, DCF conversion kinetics, mineralization of organic content, biodegradability, and toxicity were monitored and compared. The results showed different kinetics of DCF conversion regarding the type of UV source applied. However, in both cases, the mineralization extent reached upon complete DCF conversion is rather low (≤10%), suggesting that the majority of DCF was transformed into by-products. Formation/degradation of main degradation by-products was monitored using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS), whereas different profiles were obtained by UV-C and UV-A photolysis. The results of bioassays revealed that biodegradability of DCF solutions remained low through the applied treatments. The toxicity of irradiated DCF solutions was evaluated using Vibrio fischeri. A significant reduction of toxicity, especially in the case of UV-A radiation, was observed upon complete degradation of DCF. In addition to toxicity reduction, calculated Log K OW values of DCF degradation by-products indicate their low potential for bioaccumulation (Log K OW ≤ 3) in comparison to the parent substance.

Pharmacokinetics of isoforskolin after administration via different routes in guinea pigs

1. The objective of this study was to characterize the pharmacokinetics of isoforskolin after oral, intraperitoneal and intravenous administration, as well as to compare bioavailability.2. Isoforskolin was administered to guinea pigs at a dose of 2 mg/kg. Plasma concentrations were determined by high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS) method. The pharmacokinetic parameters were calculated by a noncompartmental method. A compartment model was also adopted to describe the pharmacokinetic profiles.3. The pharmacokinetic behavior of intravenously administered isoforskolin was characterized by rapid and extensive distribution (Vz = 16.82 ± 8.42 L/kg) followed by rapid elimination from the body (Cl = 9.63 ± 4.21 L/kg/h). After intraperitoneal administration, isoforskolin was absorbed rapidly (Tmax = 0.12 ± 0.05 h). The pharmacokinetic profiles of isoforskolin were similar after intraperitoneal and intravenous administration, except for the concentrations at the initial sampling times. Isoforskolin was also absorbed rapidly following oral dosing; however, the concentration–time data were best fit to a one-compartment model, which was different from that observed after intravenous and intraperitoneal administration. Following intraperitoneal and oral administration, the absolute bioavailability of isoforskolin was 64.12% and 49.25%, respectively.4. Isoforskolin is a good candidate for oral administration because of its good oral bioavailability.

Simultaneous quantification of acetaminophen and five acetaminophen metabolites in human plasma and urine by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: Method validation and application to a neonatal pharmacokinetic study.

Drug metabolism plays a key role in acetaminophen (paracetamol)-induced hepatotoxicity, and quantification of acetaminophen metabolites provides critical information about factors influencing susceptibility to acetaminophen-induced hepatotoxicity in clinical and experimental settings. The aims of this study were to develop, validate, and apply high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) methods for simultaneous quantification of acetaminophen, acetaminophen-glucuronide, acetaminophen-sulfate, acetaminophen-glutathione, acetaminophen-cysteine, and acetaminophen-N-acetylcysteine in small volumes of human plasma and urine. In the reported procedures, acetaminophen-d4 and acetaminophen-d3-sulfate were utilized as internal standards (IS). Analytes and IS were recovered from human plasma (10μL) by protein precipitation with acetonitrile. Human urine (10μL) was prepared by fortification with IS followed only by sample dilution. Calibration concentration ranges were tailored to literature values for each analyte in each biological matrix. Prepared samples from plasma and urine were analyzed under the same HPLC-ESI-MS/MS conditions, and chromatographic separation was achieved through use of an Agilent Poroshell 120 EC-C18 column with a 20-min run time per injected sample. The analytes could be accurately and precisely quantified over 2.0-3.5 orders of magnitude. Across both matrices, mean intra- and inter-assay accuracies ranged from 85% to 112%, and intra- and inter-assay imprecision did not exceed 15%. Validation experiments included tests for specificity, recovery and ionization efficiency, inter-individual variability in matrix effects, stock solution stability, and sample stability under a variety of storage and handling conditions (room temperature, freezer, freeze-thaw, and post-preparative). The utility and suitability of the reported procedures were illustrated by analysis of pharmacokinetic samples collected from neonates receiving intravenous acetaminophen.

A sensitive LC–ESI-MS/MS method for the quantification of avobenzone in rat plasma and skin layers: Application to a topical administration study

This study describes the development of a sensitive high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of avobenzone in rat plasma and skin layers. Separations were performed on a Zorbax SB C8 column using a binary gradient mobile phase composed of acetonitrile and 0.1% formic acid in water. The assay achieved LLOQ of 0.5ng/ml for plasma, 5ng/ml for stratum corneum, and 10ng/ml for epidermis and dermis. This method was applied to a percutaneous absorption study of avobenzone in rats. At 12h following topical application of emulsion and lotion (applied amount of avobenzone 11.7mg/kg), avobenzone was found primarily in the stratum corneum (16.3-17.8%) followed by epidermis (2.0-3.4%) and dermis (0.11-0.15%). Avobenzone was not quantifiable in the plasma samples collected over a 12h sampling period. Given the excellent plasma assay sensitivity, this study provides evidence that the systemic absorption of avobenzone is insignificant, if any, after topical application.

Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate.

IntroductionDNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results.MethodFive major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR).ResultsDisease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression.ConclusionsOur results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0748-5) contains supplementary material, which is available to authorized users.

Quantification of a biomarker of acetaminophen protein adducts in human serum by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: clinical and animal model applications.

The aims of this study were to develop, validate, and apply a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantification of protein-derived 3-(cystein-S-yl)-acetaminophen (APAP-Cys) in human serum. Formation of acetaminophen (APAP) protein adducts is thought to be a critical, early event in the development of APAP-induced hepatotoxicity, and quantification of these protein adducts in human serum represents a valuable tool for assessment of APAP exposure, metabolism, and toxicity. In the reported procedure, serum samples were first dialyzed or passed through gel filtration columns to remove APAP-Cys not covalently bound to proteins. Serum eluates were then subjected to enzymatic protease digestion to liberate protein-bound APAP-Cys. Norbuprenorphine-D3 was utilized as an internal standard (IS). APAP-Cys and IS were recovered from digested serum by protein precipitation with acetonitrile, and sample extracts were analyzed by HPLC-ESI-MS/MS. The method was validated by assessment of intra- and inter-assay accuracy and imprecision on two different analytical instrument platforms. APAP-Cys could be accurately quantified from 0.010 to 10μM, and intra- and inter-assay imprecision were <15% on both analytical instruments. APAP-Cys was stable in human serum for three freeze-thaw cycles and for 24h at ambient temperature. Extracted samples were stable when stored in refrigerated autosamplers for the typical duration of analysis or when stored at -20°C for six days. Results from process efficiency and matrix effect experiments indicated adequate recovery from human serum and insignificant ion suppression or enhancement. The utility and sensitivity of the reported procedure were illustrated by analysis of clinical samples collected from subjects taking chronic, therapeutic doses of APAP. Applicability to other biological matrices was also demonstrated by measurement of protein-derived APAP-Cys in plasma collected from APAP-treated mice, a common animal model of APAP-induced hepatotoxicity.

Simultaneous determination of ten alkaloids of crude and wine-processed Rhizoma Coptidis aqueous extracts in rat plasma by UHPLC–ESI–MS/MS and its application to a comparative pharmacokinetic study

Rhizoma coptidis (R.C.), a widely used traditional Chinese medicine, has been used for centuries in the treatment of hypertension, inflammation, dysentery and liver diseases, etc. Wine-processing is a specialized technology by sautéing crude herbal medicine using Chinese rice wine. This paper was designed to establish a simultaneous quantitative method of ten alkaloids (berberine, coptisine, palmatine, jatrorrhizine, epiberberine, magnoflorine, columbamine, noroxyhydrastinine, oxyberberine and 8-oxocoptisine) in rat plasma. Furthermore, the pharmacokinetics of those alkaloids after administration of crude and wine-processed R.C. aqueous extracts was compared. As a result, a ultra high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC–ESI–MS/MS) method was developed and validated for the first time. Chromatographic separation was achieved on a C18 column using gradient elution with the mobile phase consisting of acetonitrile and water (containing 0.2% formic acid) at a flow rate of 0.2ml/min. The validated method showed good linearity over a wide concentration range (r>0.99), and lower limits of quantification less than 5.46ng/ml for the each analyte. The intra- and inter-day assay variability was below 9.9% and 10.5% for all analytes, respectively. The extraction recovery of those alkaloids and I.S. ranged from 65.3% to 90.7%. The validated method has been successfully applied to pharmacokinetic comparison after administration of crude and wine-processed R.C. aqueous extracts. Pharmacokinetic comparative study showed that Cmax of coptisine and 8-oxocoptisine and AUC0−t of coptisine, palmatine and 8-oxocoptisine were increased significantly (p<0.05) after wine-processing, while other compounds didn’t show significant difference, which suggested that wine-processing exerted limited effects on the absorption of alkaloids. These results might be helpful for R.C.’ clinical reasonable application and further studies on its wine-processing mechanism.

Simultaneous quantitative determination of five alkaloids in Catharanthus roseus by HPLC-ESI-MS/MS

AimTo establish a method to simultaneously determine the main five alkaloids of Catharanthus roseus for trace samples, a high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS) analysis method was developed. MethodThe five Catharanthus alkaloids, vinblastine, vincristine, vinleurosine, vindoline, and catharanthine were chromatographically separated on a C18 HPLC column. The mobile phase was methanol–15 nmol·L−1 ammonium acetate containing 0.02% formic acid (65 : 35, V/V). The quantification of these alkaloids was based on the Multiple Reaction Monitoring (MRM) mode. ResultsThis method was validated, and the results achieved the aims of the study. The intra- and inter-day precision and accuracy of the five alkaloids were within 1.2%–11.5% (RSD%) and −10.9%–10.5% (RE%). The recovery rates of the five alkaloids of samples were from 79.9% to 91.5%. The five analytes were stable at room temperature for 2 h, at 4 °C for 12 h, and at −20 °C for two weeks. The developed method was applied successfully to determine the content of the five alkaloids in three plant parts of three batches of C. roseus with a minute amount collected from three regions of China. ConclusionThe HPLC-ESI-MS/MS method can be used for the simultaneous determination of five important alkaloids in trace C. roseus samples.

Accumulation and molecular regulation of anthocyanin in purple tumorous stem mustard (Brassica juncea var. tumida Tsen et Lee).

Tumorous stem mustard (Brassica juncea var. tumida Tsen et Lee) is an economically and nutritionally important dietary vegetable in Asian countries. Purple tumorous stem mustard contains inflated tumorous stem and abundant anthocyanin accumulation in leaves. Here, 20 anthocyanins were separated and identified from the purple tumorous stem mustard by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). In order to investigate the regulatory anthocyanin production in purple tumorous stem mustard, the expression of anthocyanin biosynthetic and regulatory genes in leaves from purple and green cultivars were examined. Regulatory gene BjTT8 and all biosynthetic genes were dramatically upregulated in the purple variety. Moreover, the transcript level of BjTT8 and all structural genes, except BjPAL, were all significantly higher in light-treated sprouts than in the dark. These results indicate that transcriptional activation of BjTT8 is associated with upregulation of most anthocyanin biosynthetic genes, to produce anthocyanins in purple tumorous stem mustard.