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.

A systematic evaluation of the cucurbit[7]uril pharmacokinetics and toxicity after a single dose and short-term repeated administration in mice.

Cucurbit[n]urils are macrocyclic compounds capable of forming host-guest complexes with different molecules. In this study, we focused on cucurbit[7]uril (CB[7]) safety and pharmacokinetics. We investigated CB[7] cytotocixity in human renal cells ACHN using the xCELLigence system. We also determined maximum tolerated doses (MTD) and no observed adverse effect levels (NOAEL) after intramuscular (i.m.), intraperitoneal (i.p.), and intragastric (i.g.) administration in mice using clinical observation, blood biochemistry, and histopathology. At NOAELs, we studied its pharmacokinetics in plasma and kidneys. Finally, we performed a 7 day repeated-dose toxicity study at 50% of NOAEL after i.p. administration, assaying CB[7] concentration in plasma, brain, kidney, and liver; we also assessed the liver and kidney histopathology. In vitro, CB[7] did not show toxicity up to 0.94 mg/mL. MTDs in vivo were set at 300, 350, and 600 mg/kg, and NOAEL were established at 150, 100, and 300 mg/kg after i.m., i.p., and i.g. administration, respectively. Parenteral administration produced tissue damage mainly to the kidney, while i.g. administration caused only minor liver damage. Parenteral CB[7] administration led to fast elimination from blood, accompanied with kidney accumulation; absorption from the gastrointestinal tract was minimal. Short repeated i.p. administration was well tolerated. After initial CB[7] accumulation in blood and kidney, the concentrations stabilised and decreased during the experiment. Approximately 3.6% of animals showed signs of nephrotoxicity. Although CB[7] appears to be a promising molecule, nephrotoxicity may be the most critical drawback of its parenteral use, because the kidney represents the main organ of its elimination.

Synthesis and biological evaluation of new N-benzylpyridinium-based benzoheterocycles as potential anti-Alzheimer's agents.

A novel series of benzylpyridinium-based benzoheterocycles (benzimidazole, benzoxazole or benzothiazole) were designed as potent acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. The title compounds 4a-q were conveniently synthesized via condensation reaction of 1,2-phenylenediamine, 2-aminophenol or 2-aminothiophenol with pyridin-4-carbalehyde, followed by N-benzylation using various benzyl halides. The results of in vitro biological assays revealed that most of them, especially 4c and 4g, had potent anticholinesterase activity comparable or more potent than reference drug, donepezil. The kinetic study demonstrated that the representative compound 4c inhibits AChE in competitive manner. According to the ligand-enzyme docking simulation, compound 4c occupied the active site at the vicinity of catalytic triad. The compounds 4c and 4g were found to be inhibitors of Aß self-aggregation as well as AChE-induced Aß aggregation. Meanwhile, these compounds could significantly protect PC12 cells against H2O2-induced injury and showed no toxicity against HepG2 cells. As multi-targeted structures, compounds 4c and 4g could be considered as promising candidate for further lead developments to treat Alzheimer's disease.

Synthesis of novel hetero ring fused pyridine derivatives; Their anticancer activity, CoMFA and CoMSIA studies.

A series of novel furo[2,3-b]pyridine-2-carboxamide 4a-h/pyrido[3',2':4,5]furo[3,2-d] pyrimidin-4(3H)-one derivatives 5a-p were prepared from pyridin 2(1H) one 1 via selective O-alkylation with α-bromoethylester followed by cyclization, then reaction with different aliphatic primary amines to obtain 4 and further reaction with triethyl orthoacetate/triethyl orthoformate. Also prepared novel furo[2,3-b]pyridine-2-carbohydrazide Schiff's bases 7a-h and pyrido [3',2':4,5]furo[3,2-d]pyrimidin-4(3H)-one derivatives 8a-h starting from furo[2,3-b]pyridine carboxylate derivatives 3 by reaction with hydrazine hydrate to form 6 and reaction with diverse substituted aldehydes and cyclization. Products 4a-h, 5a-p, 7a-h and 8a-h were screened against four human cancer cell lines (HeLa, COLO205, Hep G2 and MCF 7) and one normal cell line (HEK 293). Compounds 4e, 4f, 4g, 5h, 7c, 7d, 7e and 7f showed significant anticancer activity against all the cell lines at micro molar concentration and found to be non-toxic to normal cell line. Studies for HeLa, COLO205 and MCF-7 using CoMFA and CoMSIA. Models from 3D-QSAR provided a strong basis for future rational design of more active and selective HeLa, COLO205 and MCF-7 cell line inhibitors.

Miniaturization of the Clonogenic Assay Using Confluence Measurement.

The clonogenic assay is a widely used method to study the ability of cells to 'infinitely' produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth.

The discovery of fluazaindolizine: A new product for the control of plant parasitic nematodes.

Fluazaindolizine is a new highly effective and selective product for the control of plant parasitic nematodes. Specificity for nematodes coupled with absence of activity against the target sites of commercial nematicides suggests that fluazaindolizine has a novel mode of action. The discovery, structure-activity development and biological properties for this new class of nematicides are presented.

VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident.

Synthesis, antitumor evaluation and molecular docking studies of [1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine derivatives.

A series of [1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine derivatives have been synthesized and evaluated for their antitumor activities. These compounds exhibited potent antiproliferative activities against MCF-7, Bewo and HL-60 cells and c-Met kinase inhibitory activities. Three compounds were highly effective against MCF-7, Bewo and HL-60 cells with IC50 values in 1.09-2.24µM. Molecular docking was further performed to study the inhibitor-c-Met kinase interactions, and the results show that compound 4j was potently bound to the c-Met kinase with three hydrogen bonds. The further research on acute toxicity and in vivo antitumor activity of compound 4j to ICR (Institute of Cancer Research) mice were carried out, and found 4j with a certain toxicity but good efficacy in vivo. Based on the preliminary results, it is deduced that compound 4j with potent c-Met kinase inhibitory activity may be a potential anticancer agent.

Cross-resistance patterns to ACCase-inhibitors in American sloughgrass (Beckmannia syzigachne Steud.) homozygous for specific ACCase mutations.

American sloughgrass is a troublesome annual grass weed in winter wheat field rotated with rice in China. The overreliance on acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides has resulted in resistance evolution in this weed. In this study, the cross-resistance patterns to fenoxaprop-p-ethyl, clodinafop-propargyl, fluazifop-p-butyl, haloxyfop-p-methyl, sethoxydim, clethodim and pinoxaden were established using purified plants individually homozygous for specific mutant ACCase alleles. Results indicated that 1781Leu allele endows high-level resistance to APPs, CHDs and pinoxaden while confers moderate resistance to haloxyfop-p-methyl. The 2027Cys and 2041Asn alleles endow high-level resistance to APPs and pinoxaden and lower level resistance to CHDs. The 2078Gly allele confers high-level resistance to all herbicides tested in this study, however, moderate resistance to sethoxydim. The 2096Ala very likely endows high-level resistance to fluazifop-p-butyl, haloxyfop-p-methyl and moderate resistance to sethoxydim. In addition, one undefined resistance mechanism was involved in population SD-04.

Multiple resistance to ACCase and AHAS-inhibiting herbicides in shortawn foxtail (Alopecurus aequalis Sobol.) from China.

Shortawn foxtail (Alopecurus aequalis) is a troublesome grass weed infesting winter wheat and oilseed rape productions in China. Fenoxaprop-p-ethyl and mesosulfuron-methyl failed to control shortawn foxtail of AHSX-1 population collected from a wheat field in Shou County, Anhui province. Molecular analyses revealed that Asp2078Gly mutation of ACCase and Trp574Leu mutation of AHAS were present in plants of the AHSX-1 population. The homozygous plants were isolated and cultured until seed maturity. Whole-plant herbicide bioassays were conducted in the greenhouse using the purified seeds of F1 generation. Dose-response experiments showed that the AHSX-1 population has evolved a very high level resistance to fenoxaprop-p-ethyl (RI = 275) and mesosulfuron-methyl (RI = 788). To determine the sensitivity to other herbicides, assays were conducted at the single recommended rate of each herbicide. Based on the results, the AHSX-1 population was considered to be highly resistant to clodinafop-propargyl, pyroxsulam and flucarbazone-sodium, moderately or highly resistant to quizalofop-p-ethyl, clethodim, sethoxydim and pinoxaden, and susceptible to isoproturon and chlorotoluron. This is the first report of Asp2078Gly mutation in shortawn foxtail and the two robust dCAPS markers designed could quickly detect Asp2078 and Trp574 mutations in ACCase and AHAS gene of shortawn foxtail, respectively.

Resistance of American sloughgrass (Bechmannia syzigachne) populations to ACCase-inhibiting herbicides involves three different target site mutations from China.

American sloughgrass [Beckmannia syzigachne (Steud.) Fernald] is a problematic annual grass weed in winter wheat fields of China, which causes great loss of wheat yield. Repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides during the last two decades to control this weed has been selected for resistance in American sloughgrass in Jiangsu province. In this study, whole-plant dose-response assays were conducted to investigate the level of resistance in four resistant American sloughgrass populations (LY, JH, BYJ and BYP) to four ACCase-inhibiting herbicides belonging to aryloxyphenoxypropionates, cyclohexanediones, and phenylpyrazolines groups under greenhouse conditions. Based on resistance factor (RF), three populations, LY, BYJ and BYP, were highly resistant to fenoxaprop-P-ethyl, clodinafop propargyl, sethoxydim and pinoxaden. JH plants exhibited resistance to fenoxaprop-P-ethyl and clodinafop propargyl, but showed much lower RF values for sethoxydim and pinoxaden. Molecular analysis of resistance revealed that resistance in all the four populations was target site-based. Results confirmed that substitutions of Ile-1781-Leu, Ile-2041-Asn and Asp-2078-Gly, respectively, in LY, JH and BYJ/BYP, are responsible for diverse sensitivity to different ACCase-inhibiting herbicides in these populations. The substitution at position 1781 had been reported, while it is the first report of Ile-2041-Asn and Asp-2078-Gly mutations that corresponded to resistance in American sloughgrass.

Identification of pyrroloformamide as a cytokinesis modulator.

Discovered in the late 1940s, the pyrrolinonodithioles represent a family of potent disulfide-containing natural products. Although they are understood in a synthetic and biosynthetic context, the biological role of these materials remains unresolved. To date, their activity has been suggested to arise through regulating RNA metabolism, and more recently they have been suggested to function as backup thiols for detoxification. Using materials identified through a natural products program, we now identify the biological function of one member of this family, pyrroloformamide, as an antimitotic agent acting, in part, by disrupting cytokinesis.

Target-site mechanism of ACCase-inhibitors resistance in American sloughgrass (Beckmannia syzigachne Steud.) from China.

American sloughgrass (Beckmannia syzigachne) is a troublesome weed in winter wheat field rotated with rice in China. Fenoxaprop-p-ethyl and pinoxaden were observed failing to control American sloughgrass in the same filed in Lujiang county in 2011 and 2012, respectively. Whole-plant bioassay was conducted to determine the resistance to fenoxaprop-p-ethyl, pinoxaden and other herbicides in American sloughgrass. Dose-response experiment indicated that Lujiang population was highly resistant to fenoxaprop-p-ethyl (199.8-fold), pinoxaden (76.2-fold), clodinafop-propargyl (334.1-fold) and sethoxydim (15.9-fold); moderately resistant to clethodim (6.3-fold), susceptible to mesosulfuron-methyl, flucarbazone-sodium, pyroxsulam and isoproturon. Partial gene of CT domain was cloned and sequenced to confirm the molecular mechanism of resistance to ACCase-inhibiting herbicides. A Trp2027Cys mutation was found in Lujiang population according to the sequencing result. This mutation is the molecular mechanism of resistance to fenoxaprop-p-ethyl in Lujiang population. Furthermore the Trp2027Cys mutation very likely results in cross resistance to clodinafop-propargyl and pinoxaden in Lujiang population. 103 mutant homozygotes were detected from the 108 plants tested using a rapid dCAPS method developed in this paper. This is the first report of pinoxaden resistance and a mutation at position of 2027 for American sloughgrass.

Differential effects of crambescins and crambescidin 816 in voltage-gated sodium, potassium and calcium channels in neurons.

Crambescins and crambescidins are two families of guanidine alkaloids from the marine sponge Crambe crambe. Although very little information about their biological effect has been reported, it is known that crambescidin 816 (Cramb816) blocks calcium channels in a neuroblastoma X glioma cell line. Taking this into account, and the fact that ion channels are frequent targets for natural toxins, we examined the effect of Cramb816 and three compounds from the crambescin family, norcrambescin A2 (NcrambA2), crambescin A2 (CrambA2), and crambescin C1 (CrambC1), in the main voltage-dependent ion channels in neurons: sodium, potassium, and calcium channels. Electrophysiological recordings of voltage gated sodium, potassium, and calcium currents, in the presence of these guanidine alkaloids, were performed in cortical neurons from embryonic mice. Different effects were discovered: crambescins inhibited K(+) currents with the following potency: NcrambA2 > CrambC1 > CrambA2, while Cramb816 lacked an effect. Only CrambC1 and Cramb816 partially blocked Na(+) total current. However, Cramb816 partially blocked Ca(2+) , while NcrambA2 did not. Since the blocking effect of Cramb816 on calcium currents has not been previously reported in detail, we further pharmacologically isolated the two main fractions of HVA Ca(2+) channels in neurons and investigated the Cramb816 effect on them. Here, we revealed that Cav1 or L-type calcium channels are the main target for Cramb816. These two families of guanidine alkaloids clearly showed a structure-activity relationship with the crambescins acting on voltage-gated potassium channels, while Cramb816 blocks the voltage-gated calcium channel Cav1 with higher potency than nifedipine. The novel evidence that Cramb816 partially blocked CaV and NaV channels in neurons suggests that this compound might be involved in decreasing the neurotransmitter release and synaptic transmission in the central nervous system. The findings presented here provide the first detailed approach on the different effects of crambescin and crambescidin compounds in voltage-gated sodium, potassium, and calcium channels in neurons and thus provide a basis for future studies.

Mitigation of cardiovascular toxicity in a series of CSF-1R inhibitors, and the identification of AZD7507.

The potent and selective 3-amido-4-anilinoquinoline CSF-1R inhibitor AZ683 suffered from cardiovascular liabilities, which were linked to the off-target activities of the compound and ion channel activity in particular. Less basic and less lipophilic examples from both the quinoline and cinnoline series demonstrated cleaner secondary pharmacology profiles. Cinnoline 31 retained the required potency and oral PK profile, and was progressed through the safety screening cascade to be nominated into development as AZD7507.

Furanodiene, a natural product, inhibits breast cancer growth both in vitro and in vivo.

PURPOSE: Previous studies have reported that the Curcuma wenyujin Y.H. Chen et C. Ling extract, which has a high furanodiene content, showed anti-cancer effects in breast cancer cells in vitro. The present study was designed to evaluate the in vitro and in vivo anti-cancer activity of furanodiene. METHODS: The in vitro effects of furanodiene were examined on two human breast cancer cell lines, MCF-7 and MDA-MB-231 cells. Assays of proliferation, LDH release, mitochondrial membrane potential (ΔΨm), cell cycle distribution, apoptosis and relevant signaling pathways were performed. The in vivo effect was determined with MCF7 tumor xenograft model in nude mice. RESULTS: Furanodiene significantly inhibited the proliferation and increased the LDH release in both cell lines in a dose-dependent manner. ΔΨm depolarization, chromatin condensation, and DNA fragmentation were also observed after furanodiene treatment. Furanodiene dose-dependently induced cell cycle arrest at the G0/G1 phase. The protein expressions of p-cyclin D1, total cyclin D1, p-CDK2, total CDK2, p-Rb, total Rb, Bcl-xL, and Akt were significantly inhibited by furanodiene, whereas the protein expressions of Bad and Bax, and the proteolytic cleavage of caspase-9, caspase-7, and poly-ADP-ribose polymerase (PARP) were dramatically increased. Furthermore, the z-VAD-fmk markedly reversed the furanodiene-induced cell cytotoxicity, the proteolytic cleavage of caspase-9, and DNA fragmentation but did not affect the proteolytic cleavage of PARP, whereas the Akt inhibitor VIII increased the furanodiene-induced cytotoxicity and PARP cleavage. In addition, furanodiene dose-dependently suppressed the tumor growth in vivo, achieving 32% and 54% inhibition rates after intraperitoneal injection of 15 mg/kg and 30 mg/kg, respectively. CONCLUSIONS: Taken together, we concluded that furanodiene suppresses breast cancer cell growth both in vitro and in vivo and could be a new lead compound for breast cancer chemotherapy.

Cyto-genotoxic effects of Pinoxaden on Allium cepa L. roots.

Pinoxaden is the one of the acetyl-CoA carboxylases (ACCase) inhibiting herbicides and used for controlling grass weeds. In this study, cyto-genotoxic effects of Pinoxaden on the Allium cepa roots were investigated using Allium ana-telophase and comet assays by determining the root growth, mitotic index (MI), mitotic phases, chromosomal aberrations (CAs) and DNA damage. Different concentrations of Pinoxaden from 0.5 to 100 mg/L were employed on root tips for 96 h to find the effective concentration that reduces root tip elongation by 50% in comparison with negative control (EC50). Pinoxaden concentrations of 1.25 mg/L (1/2xEC50), 2.5 mg/L (EC50) and 5 mg/L (2xEC50); methyl methane sulphonate (MMS, 10 mg/L) for positive control and distilled water for negative control were exposed to Allium bulbs for several time intervals (24, 48, 72 and 96 h). Pinoxaden showed cytotoxic effects by decreasing the root growth and MI. Pinoxaden induced CAs including disturbed ana-telophase, anaphase bridges, chromosome laggards, stickiness, polyploidy, micronucleus at 5 mg/L, c-metaphase and binuclear cells and also DNA damage compared with control group. The current study confirmed cyto-genotoxic effects of Pinoxaden. Further research is needed to clarify the cyto-genotoxic mechanisms of Pinoxaden at molecular level.

Incarviditone: a novel cytotoxic benzofuranone dimer from Incarvillea delavayi Bureau et Franchet.

A novel benzofuranone dimer, named incarviditone (1), along with known rengyolone (2), was isolated from Incarvillea delavayi Bureau et Franchet. The structure of the new compound was elucidated on the basis of spectroscopic methods, especially 2D-NMR and MS analyses. Compound 1 is the first example of a benzofuranone dimer connected by a C--C bond, which presents a new C-skeleton. The cytotoxicities of compounds 1 and 2 were evaluated against the cell lines A549, LOVO, HL-60, 6T-CEM, and HepG2. Incarviditone (1) exhibited cytotoxicity only against HL-60 and 6T-CEM cell lines with IC(50) values of 14.8 and 22.2 microg/ml, respectively, while rengyolone (2) was more active against all cell lines with IC(50) values between 6.7 and 20.2 microg/ml.

Black-grass resistance to herbicides: three years of monitoring in Belgium.

Black-grass (Alopecurus myosuroides HUDS.) is a common weed of cereal crops widely spread in Northern Europe. Even if the first Belgian case of resistance was reported in 1996, until now, Belgium was quite spared of this problem and only a few restricted areas were concerned: the Polders, the marshland of the Escaut River and the Fosses-la-Ville region. About 90 seed samples were collected trough the South part of Belgium and in the Polders during July 2006, 2007 and 2008. These populations were tested in greenhouse conditions by spraying plantlets with herbicides of three modes of action. The herbicides used were photosynthesis inhibitor, ACCase inhibitors and ALS inhibitors. Susceptible and resistant standard populations (Rothamsted and Peldon) were included in the test in order to validate it and to permit wild populations classification according to "R" rating system. Populations showed differences of susceptibility to photosynthesis inhibitor, ACCase inhibitors and ALS inhibitors. For each herbicide mode of action, it was possible to find at least one population in each resistance class of the "R" rating system. Furthermore, it appeared that resistance was not confined to restricted areas listed above anymore.

A Simple LC-MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass.

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography-mass spectrometry (LC-MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC-MS (for peramine). This work provides the first highly sensitive quantitative LC-MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.