Analysis of omarigliptin forced degradation products by ultra-fast liquid chromatography, mass spectrometry, and in vitro toxicity assay.

Omarigliptin (OMG) is an antidiabetic drug indicated for the treatment of type 2 diabetes mellitus. Forced degradation studies are practical experiments to evaluate the stability of drugs and to establish degradation profiles. Herein, we present the investigation of the degradation products (DPs) of OMG formed under various stress conditions. OMG was subjected to hydrolytic (alkaline and acidic), oxidative, thermal, and photolytic forced degradation. A stability-indicating ultra-fast liquid chromatography method was applied to separate and quantify OMG and its DPs. Five DPs were adequately separated and detected in less than 6 min, while other published methods detected four DPs. MS was applied to identify and obtain information on the structural elucidation of the DPs. Three m/z DPs confirmed previously published research, and two novel DPs were described in this paper. The toxicity of OMG and its DPs were investigated for the first time using in vitro cytotoxicity assays, and the sample under oxidative conditions presented significant cytotoxicity. Based on the results from forced degradation studies, OMG was found to be labile to hydrolysis, oxidation, photolytic, and thermal stress conditions. The results of this study contribute to the quality control and stability profile of OMG.

The Difference of Volatile Compounds in Female and Male Buds of Trichosanthes anguina L. Based on HS-SPME-GC-MS and Multivariate Statistical Analysis.

Trichosanthes anguina L. (family Cucurbitaceae) is a monoecious and diclinous plant that can be consumed as a vegetable and has anti-inflammatory and antioxidant effects. The chemical composition and content of volatile compounds in female and male buds of T. anguina were explored by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology combined with multivariate statistical analysis. The results showed that the content of the volatile compounds was different between female and male buds. 2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol and 2,2,6-trimethyl-6-vinyldihydro-2H-pyran-3(4H)-one were the main volatile compounds in both female and male buds. Based on the multivariate statistical analysis of orthogonal projections to latent structures discriminant analysis (OPLS-DA) and t-test, the content of seven compounds was significantly different between female and male buds. The content of three compounds in male buds was higher than that in female, i.e., (E)-4,8-dimethyl-1,3,7-nonatriene, 1,5,9,9-tetramethyl-1,4,7-cycloundecatriene, and (E)-caryophyllene. Conversely, the content of (Z)-4-hexen-1-ol, (Z)-3-hexenyl benzoate, (Z)-3-hexenyl salicylate, and 2-hexen-1-ol in female buds was higher than that in male buds. This is the first report on the difference in the volatile compounds between female and male buds of T. anguina, which enriches the basic research on the monoecious and diclinous plant and provides a reference for the study of plant sex differentiation.

Establishing an efficient salinomycin biosynthetic pathway in three heterologous Streptomyces hosts by constructing a 106-kb multioperon artificial gene cluster.

Salinomycin is a promising anticancer drug for chemotherapy. A highly productive biosynthetic gene cluster will facilitate the creation of analogs with improved therapeutic activity and reduced side effects. In this study, we engineered an artificial 106-kb salinomycin gene cluster and achieved efficient heterologous expression in three hosts: Streptomyces coelicolor CH999, S. lividans K4-114, and S. albus J1074. The six-operon artificial gene cluster consists of 25 genes from the native gene cluster organized into five operons and five fatty acid ß-oxidation genes into one operon. All operons are driven by strong constitutive promoters. For K4-114 and J1074 harboring the artificial gene cluster, salinomycin production in shake flask cultures was 14.3 mg L-1 and 19.3 mg L-1 , respectively. The production was 1.3-fold and 1.7-fold higher, respectively, than that of the native producer S. albus DSM41398. K4-114 and J1074 harboring the native gene cluster produced an undetectable amount of salinomycin and 0.5 mg L-1 , respectively. CH999 harboring the artificial gene cluster produced 10.3 mg L-1 of salinomycin, which was 92% of the production by DSM41398. The efficient heterologous expression system based on the 106-kb multioperon artificial gene cluster established in this study will facilitate structural diversification of salinomycin, which is valuable for drug development and structure-activity studies.

Detection of salinomycin and lasalocid in chicken liver by icELISA based on functional bispecific single-chain antibody (scDb) and interpretation of molecular recognition mechanism.

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9-24.0 and 4.7-16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2-92.7%(CV<4.7%) and 88.6-90.2% (CV<6.8%)), respectively.

Salinomycin-loaded PLA nanoparticles: drug quantification by GPC and wave voltammetry and biological studies on osteosarcoma cancer stem cells.

A new straightforward gel permeation chromatography (GPC) method was developed to calculate the drug encapsulation efficiency and loading content of Poly(lactic acid) nanoparticles (PLA NPs) loaded with Salinomycin (Sal), exploiting the capability of this technique to separate a macromolecular/molecular mixture on the basis of the molecular weight of each component. The proposed GPC method allowed Sal detection until 1% of Sal content in PLA NPs, avoiding sample pre-treatments. The method was validated by wave voltammetry (SW) technique, using a slightly modified literature procedure, useful to detect Sal in the concentration range 0.4 ≤ C/µmol/L ≤ 12 (linear concentration range). PLA-based NPs were prepared by nanoprecipitation with either native and functionalized PLA. Specifically, folate-decorated PLA NPs (PLA-FA NPs) were obtained by CuAAC click functionalization of alkyne-grafted PLA with azide-folate. Sal-loaded NPs were characterized physicochemically and morphologically. They exhibited adequate physicochemical properties, good drug encapsulation efficiency (98 ± 0.5% and 99 ± 0.5%), and loading content (8.8 ± 0.1% and 8.9 ± 0.1% for PLA/Sal and PLA-FA/Sal NPs, respectively). The size of empty PLA NPs resulted smaller (90 ± 3.2 nm and 680 ± 15.3 nm, for PLA NPs and PLA-FA NPs respectively) than the correspondent drug-loaded NPs (110 ± 3.8 nm and 875 ± 20.5 nm, respectively). Their biological activity was assessed on osteosarcoma bulk cells MG63, healthy osteoblast cell line (hFOB1.19), and enriched osteosarcoma cancer stem cells (CSCs), showing cell-depending effect. Entrapped Sal maintained its cytotoxic effect on CSCs and MG63 cells, with a potency comparable to the free drug and no evident benefit was detected for folate-decorated PLA NPs respect to native PLA NPs. Graphical abstract.

Identification of [6-Hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2 H-pyran-3-yl]-cysteine (HHPC) as a Cysteine-specific Modification Formed from 3,4-Dideoxyglucosone-3-ene (3,4-DGE).

Glucose degradation products (GDPs) are formed from glucose and other reducing sugars during heat treatment, for example, in heat-sterilized peritoneal dialysis fluids or foods. Because of their reactive mono- and dicarbonyl structure, they react readily with proteins, resulting in the formation of advanced glycation end products (AGEs), loss of protein functionality, and cytotoxicity. Among the GDPs, 3,4-dideoxyglucosone-3-ene (3,4-DGE) exerts the strongest effects despite its relatively low concentration levels. The goal of the present study was therefore to identify the structure of specific protein modifications deriving from 3,4-DGE. A nonapeptide containing the reactive amino acids lysine, arginine, and cysteine was incubated with 3,4-DGE and the dominant GDPs 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal) in concentrations as present in peritoneal dialysis fluids (235 µM 3-DG, 100 µM 3-Gal, and 11 µM 3,4-DGE). Glycation rate and product formation were determined by ultra-HPLC-MS/MS (UHPLC-MS/MS). 3,4-DGE showed the strongest glycation activity. After 2 h of incubation, 3,4-DGE had modified 57% of the nonapeptide, whereas 3-DG had modified only 2% and 3-DGal had modified 29% of the peptide. A stable 3,4-DGE-derived cysteine modification was isolated. Its structure was determined by comprehensive NMR and MS experiments to be [6-hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2 H-pyran-3-yl]-cysteine (HHPC), which represents a novel cysteine-AGE derived from 3,4-DGE. The results indicate that 3,4-DGE might contribute to a severe loss of protein functionality by forming cysteine-specific AGEs, such as HHPC.

Infrared-assisted extraction followed by high performance liquid chromatography to determine angoroside C, cinnamic acid, and harpagoside content in Scrophularia ningpoensis.

BACKGROUND: Angoroside C, cinnamic acid, and harpagoside are bioactive constituents in Scrophularia ningpoensis. Currently, an infrared-assisted extraction (IRAE) method coupled with high-performance liquid chromatography with ultraviolet detection (HPLC-UV) for the analysis of bioactive constituents in this plant is lacking. METHODS: A method based on HPLC following IRAE has been developed for quantifying angoroside C, cinnamic acid, and harpagoside in Scrophularia ningpoensis. Four main factors, namely, extraction solvent, solid/liquid ratio, illumination time, and distance between the infrared lamp and the round-bottom flask, were optimized for extraction. Furthermore, conventional ultrasonic extraction (USE) and microwave-assisted extraction (MAE) were also investigated to validate the developed method. RESULTS: The optimal extraction conditions were as follows: ethanol concentration, 37.5%; solid/liquid ratio, 1:25; illumination time, 10 min; and distance between infrared lamp and round-bottom flask, 3 cm. The results of method validation demonstrated that the developed method meets the requirement of analysis. CONCLUSION: The results show that the IRAE-HPLC is a simple, accurate, and green analytical preparatory method for the potential extraction and quantification of angoroside C, cinnamic acid, and harpagoside in Scrophularia ningpoensis.

2H-Pyranone and isocoumarin derivatives isolated from the plant pathogenic fungus Leptosphaena maculans.

Two new 2H-pyranones and two new isocoumarin derivatives, maculanslines A-D (1-4), together with seven known compounds (5-11), were isolated from the plant pathogenic fungus Leptosphaena maculans. Their planar structures and absolute configuration were elucidated by comprehensive spectroscopic techniques including high-resolution electrospray ionization mass spectrum, 1D and 2D nuclear magnetic resonance, as well as electronic circular dichroism. All 11 compounds were tested for their inhibitory activity against α-glucosidase. Compound 1 showed moderate inhibitory activity against α-glucosidase with IC50 of 74.35 µM.

SPME-GC/MS Analysis of Methanol in Biospecimen by Derivatization with Pyran Compound.

Methanol is metabolized in the body to highly toxic formaldehyde and formate when consumed accidentally. Methanol has been typically analyzed with gas chromatography-flame ionization detector (GC-FID). However, its retention time may overlap with other volatile compounds and lead to confusion. Alternative analysis of methanol using gas chromatography/mass spectrometry (GC/MS) also has limitations due to its similar molecular weight with oxygen and low boiling point. In this study, methanol and internal standard of deuterium-substituted ethanol were derivatized with 3,4-dihydro-2H-pyran under acid catalysis using concentrated hydrochloric acid. The reaction products including 2-methoxytetrahydropyran were extracted with solid-phase microextraction followed by GC/MS analysis. This method was successfully applied to measure the lethal concentration of methanol in the blood of a victim with a standard addition method to overcome the complex matrix effect of the biospecimen. Identification of the metabolite formate by ion chromatography confirmed the death cause to be methanol poisoning. This new method was a much more convenient and reliable process to measure methanol in complex matrix samples by reducing sample pretreatment effort and cost.

Toxic Action Reevaluation of Okadaic Acid, Dinophysistoxin-1 and Dinophysistoxin-2: Toxicity Equivalency Factors Based on the Oral Toxicity Study.

BACKGROUND/AIMS: Okadaic acid (OA) and the structurally related compounds dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine phycotoxins that cause diarrheic shellfish poisoning (DSP) in humans due to ingestion of contaminated shellfish. In order to guarantee consumer protection, the regulatory authorities have defined the maximum level of DSP toxins as 160 µg OA equivalent kg-1 shellfish meat. For risk assessment and overall toxicity determination, knowledge of the relative toxicities of each analogue is required. In absence of enough information from human intoxications, oral toxicity in mice is the most reliable data for establishing Toxicity Equivalence Factors (TEFs). METHODS: Toxins were administered to mice by gavage, after that the symptomatology and mice mortality was registered over a period of 24 h. Organ damage data were collected at necropsy and transmission electron microscopy (TEM) was used for ultrastructural studies. Toxins in urine, feces and blood were analyzed by HPLC-MS/MS. The evaluation of in vitro potencies of OA, DTX1 and DTX2 was performed by the protein phosphatase 2A (PP2A) inhibition assay. RESULTS: Mice that received DSP toxins by gavage showed diarrhea as the main symptom. Those toxins caused similar gastrointestinal alterations as well as intestine ultrastructural changes. However, DSP toxins did not modify tight junctions to trigger diarrhea. They had different toxicokinetics and toxic potency. The lethal dose 50 (LD50) was 487 µg kg-1 bw for DTX1, 760 µg kg-1 bw for OA and 2262 µg kg-1 bw for DTX2. Therefore, the oral TEF values are: OA = 1, DTX1 = 1.5 and DTX2 = 0.3. CONCLUSION: This is the first comparative study of DSP toxins performed with accurate well-characterized standards and based on acute toxicity data. Results confirmed that DTX1 is more toxic than OA by oral route while DTX2 is less toxic. Hence, the current TEFs based on intraperitoneal toxicity should be modified. Also, the generally accepted toxic mode of action of this group of toxins needs to be reevaluated.

Evaluation of phenolic composition and antioxidant activity changes in olive flowers during development using HPLC/DAD and LC-MS/MS.

Olive fruit and leaves have been extensively studied for their chemical compositions and biological activities. However, less attention has been given to its flowers. The present research was achieved on Tunisian olive flowers. It aimed at studying the effects of flower development on phenolic compounds and antioxidant activity. The extracts were analyzed using high performance liquid chromatography coupled to diode array detection (HPLC/DAD) and coupled to mass spectrometry (LC-MS/MS). The HPLC/DAD analysis indicated that oleuropein aglycon (from 1.158 to 3.746 g/kg), followed by hydroxytyrosol (from 0.168 to 1.581 g/kg) and oleoside (from 0.143 to 1.325 g/kg) were the predominant phenolics in olive flowers extracts during development stages. Twenty compounds have been identified, revealing the complex profile of olive flowers, composed, in order of abundance, by secoiridoids, phenolic alcohols, lignans, flavonoids and phenolic acids. Total phenolic contents increased from 2.455 to 8.541 g/kg Gallic acid equivalent per kg of fresh flowers during all steps of the flower development. A correlation between antioxidant activity and total phenolic contents was determined.

Chemical metabolome assay by high-resolution Orbitrap mass spectrometry and assessment of associated antitumoral activity of Actinocephalus divaricatus.

RATIONALE: Actinocephalus divaricatus (Eriocaulaceae) is an important source of income for rural communities as it is sold as an ornamental plant. To date, no investigation has been conducted concerning the chemical composition and biological studies of the aerial parts of A. divaricatus. METHODS: The methanolic extract of the aerial parts of this species was chemically characterized. We applied an analytical dereplication approach based on Liquid Chromatography coupled to High-Resolution Orbitrap Mass Spectrometry in order to develop, identify and define rapidly the metabolite fingerprint of the aerial parts of A. divaricatus. Biological in vitro antitumor tests were undertaken using breast and lung cell lines of mice and humans. RESULTS: High-Resolution Mass Spectrometry (HRMS) allowed the fast determination of 30 compounds, which comprised three different classes of compounds: naphthopyranones, flavonoids and saponins. Chromatographic fractionation of the crude methanolic extract validated these results, since it led to the isolation of compounds belonging to the aforementioned classes of compounds, including new acyl glycosylated flavonoids (6-hydroxy-7-methoxyquercetin-3-O-(2"-O-acetyl)-ß-D-glucopyranoside and 6-hydroxy-7-methoxyquercetin-3-O-(6"-O-acetyl)-ß-D-glucopyranoside), which were fully characterized by Nuclear Magnetic Resonance and Mass Spectrometry experiments, and a known triterpenic saponin (3-O-ß-D-glucuronopyranosyl-30-norolean-12,20(29)-dien-28-O-ß-D-glucopyranosyl ester). Biological assays indicated that the methanolic extract of the capitula exhibited the best in vitro cytotoxicity against MCF7 cells (human breast cancer). CONCLUSIONS: The HRMS technique enabled us to identify several classes of compounds. In addition, saponins were identified for the first time in plants belonging to the Eriocaulaceae family. Thus, the essential contribution of this work lies in the new elements it brings to the taxonomic discussion which the Actinocephalus genus as a distinct genus of the Paepalanthus. The results obtained show that the methanolic extract of the capitula could be a promising source of bioactive fractions and/or compounds that may contribute towards breast cancer treatment.

Quantification of oleacein and oleuropein aglycone in olive oil using deuterated surrogates by normal-phase ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

An analytical method for the analysis of relevant secoiridoid-based components in olive oil, oleacein and oleuropein aglycone, is described using for the first time deuterated surrogates. 0.2 g of sample was necessary to perform the analysis using liquid-liquid extraction and ultrasound-assisted extraction with a mixture of methanol/water (4:1, v/v). To avoid the formation of by-products, normal-phase ultra high performance liquid chromatography was chosen for the chromatographic separation. The selected mobile phase was a gradient mixture of tetrahydrofurane and hexane, and an ACE Excel 3 CN-ES column as stationary phase. The detection and quantification was performed with a SYNAPT G2-Si mass spectrometer. The calibration curves for oleacein and oleuropein aglycone were linear and quadratic, respectively. The validation was done at three levels of concentration. Relative errors from 0.1 to 10.5% and relative standard deviations lower than 9% were obtained. The method was applied to study different samples of olive oil.

Single-Lab Validation for Determination of Kavalactones and Flavokavains in Piper methysticum (Kava).

Piper methysticum (Kava) is a plant whose roots are used in the preparation of traditional beverages with spiritual, medicinal, and social importance for the Pacific Islanders. Kava is also sold as a herbal supplement or recreational beverage consumed for its mild inebriating effect in Europe and North America. With an ongoing interest in the safety and quality of kava products, it is necessary to develop a validated method for determination of kava chemical composition to ensure confidence in quality assessment. Thus, an high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method was developed, optimized, and validated for determining six major kavalactones and three flavokavains in kava raw materials and finished products based on AOAC single-laboratory validation guidelines. This is the first fully validated analytical method for measuring kavalactones and flavokavains in a single run. The separation of the analytes was achieved in 10 min with an Agilent Poroshell C18 column using gradient separation. The sample was extracted with methanol first and then acetone. The signals were detected at 240 nm and 355 nm. The limit of quantification was under 1.2 µg/mL (0.3 mg/g) for kavalactones and under 0.35 µg/mL (0.01 mg/g) for flavokavains. The Horwitz ratio values described ranged from 0.3 to 1.82. The spike recovery experiments showed an accuracy between 92 and 105% for all analytes. The results of the study demonstrate that the method is fit for the purpose of determining methysticin, dihydromethysticin, kavain, dihydrokavain, yangonin, desmethoxyyangonin, flavokavain A, flavokavain B, and flavokavain C in kava raw material and finished products (dry-filled capsule, liquid phytocaps, and tincture).

Investigation of the in vivo metabolism of harpagoside and distribution of its metabolites in rats by HPLC-IT-TOF-MSn.

Harpagoside, an iridoid glycoside, is the major bioactive constituent of the traditional Chinese medicine Scrophulariae Radix. High-performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry (HPLC-ESI-IT-TOF-MSn ) was used to profile and identify the metabolites of harpagoside in rats in vivo and to study the distribution of these metabolites in rats for the first time. A total of 45 metabolites were identified, 37 of which were postulated to be new compounds. The number of detected metabolites in the heart, liver, spleen, lung, kidney, stomach and small intestine was 2, 9, 6, 16, 4, 16 and 6, respectively, which indicated that the target organs of harpagoside should be spleen, lung and stomach. The main types of metabolic reactions of harpagoside in rats are hydrolysis, reduction, sulfuric acid addition, hydroxylation, methoxylation, sulfate substitution, methylation, glucose conjugation and amino acid conjugation. Furthermore, 23 metabolites were determined to have bioactivities based on the literature and 'PharmMapper' analysis. These findings are useful for better comprehension of the effective forms, target organs and pharmacological effects of harpagoside. Moreover, these findings provide a reference for studying the metabolism and distribution of iridoid compounds.

Use of Innovative (Micro)Extraction Techniques to Characterise Harpagophytum procumbens Root and its Commercial Food Supplements.

INTRODUCTION: For the determination of harpagoside and the wide phenolic pattern in Harpagophytum procumbens root and its commercial food supplements, dispersive liquid-liquid microextraction (DLLME), ultrasound-assisted DLLME (UA-DLLME), and sugaring-out liquid-liquid extraction (SULLE) were tested and compared. OBJECTIVES: In order to optimise the extraction efficiency, DLLME and UA-DLLME were performed in different solvents (water and aqueous solutions of glucose, ß-cyclodextrin, (2-hydroxypropyl)-ß-cyclodextrin, sodium chloride, natural deep eutectic solvent, and ionic liquid). MATERIAL AND METHODS: The plant material was ground and sieved to obtain a uniform granulometry before extraction. Commercial food supplements, containing H. procumbens are commercially available in Italy. RESULTS: The most effective sodium chloride-aided-DLLME was then optimised and applied for analyses followed by HPLC-PDA. For comparison, microwave-assisted extraction was performed using the same solvents and the best results were obtained using 1% of ß-cyclodextrin or 15% of sodium chloride. CONCLUSION: All commercial samples respected the European Pharmacopoeia monograph for this plant material, showing a harpagoside content ≥ 1.2%. Copyright © 2017 John Wiley & Sons, Ltd.

Preparative Separation of Phenylethanoid and Secoiridoid Glycosides from Ligustri Lucidi Fructus by High-Speed Counter-Current Chromatography Coupled with Ultrahigh Pressure Extraction.

Three phenylethanoid glycosides, echinacoside (1), salidroside (3), and acteoside (6), and three secoiridoid glycosides, isonuezhenide (2), nuezhenoside G13 (4), and specnuezhenide (5), have been extracted and separated by a combined method of ultrahigh pressure extraction (UPE) and high-speed counter-current chromatography (HSCCC) from Ligustri Lucidi Fructus. For the UPE, the optimal extraction was developed with conditions including solvent of 90% ethanol, sample to solvent ratio of 1:20 g/mL, pressure of 200 MPa, and time of 2 min, which rendered the yields of compounds 4 and 5 were 15.0 and 78.0 mg/g, respectively. For the HSCCC separation, the strategy of changing flow rates between 1.0 and 2.0 mL/min allowed the acquisition for 2.7 mg of compound 1, 4.5 mg of compound 2, 6.8 mg of compound 3, 5.9 mg of compound 4, 11.2 mg of compound 5, and 2.2 mg of compound 6 in one separation run under the solvent system of ethyl acetate:n-butanol:water (2:1:3, v/v) from 200 mg of the UPE extract. The structures of these phenylethanoid and secoiridoid glycosides were elucidated by extensive spectroscopic methods.

A Comprehensive Quality Evaluation Method Based on C30-HPLC and an Analytic Hierarchy Process for the Chinese Herbal Formula, Erzhiwan.

The quantitative analysis of multiple indexes remains an important quality evaluation method of traditional Chinese medicine (TCM) herbal formulas. The Chinese Pharmacopoeia 2015 only stipulates the content of a single component, specnuezhenide, in Erzhiwan composed of the Fructus Ligustri Lucidi (FLL) powder and aqueous extracts of Herba Ecliptae (HE). To generalize the intrinsic quality of Erzhiwan, a novel C30-HPLC method with good precision, accuracy, and reproducibility was developed for the simultaneous determination of six compounds, including two isomers, and then an analytic hierarchy process was further applied to integrate and discriminate the quality of four samples prepared via different methods. The results of the analysis were in agreement with the antioxidant tests in vitro. This comprehensive strategy could provide a reference and suggestions for the improvement of the quality evaluation method of TCM herbal formulas.

A mussel tissue certified reference material for multiple phycotoxins. Part 4: certification.

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.

Quantitative Determination of Lactones in Piper methysticum (Kava-Kava) by Supercritical Fluid Chromatography.

A fast and validated supercritical fluid chromatography method for the quantitative determination of major lactones in Piper methysticum, a plant used against nervous anxiety, stress, and restlessness, was developed. The baseline separation of dihydrokavain, demethoxyyangonin, kavain, yangonin, dihydromethysticin, and methysticin was possible in less than 4 min on an Aquity UPC2 BEH 1.7 µm column, in combination with a mobile phase comprising CO2 and methanol with diethylamine. The column temperature had a great impact on the results because only at 70 °C could kavain and yangonin be fully resolved. With correlation coefficients above 0.998, recovery rates between 95.9 and 104.1 % as well as limit of detection values below 1.5 ng on-column, the procedure fulfilled all validation requirements and was well suited for the quantitative analysis of commercial products containing P. methysticum root powder and/or extract. All of them contained the target analytes, however, the absolute content of lactones was quite variable. Accordingly, depending on the product, the total daily intake of lactones varied from 56 to 312 mg. Concerning speed, selectivity, and environmental friendly operation, this supercritical fluid chromatography approach surpasses all previously reported ones.