Detection and Quantitation of Selected Food Allergens by Liquid Chromatography with Tandem Mass Spectrometry: First Action 2017.17.

Background: In response to a need for accurate and reliable methods for food allergen regulatory compliance, a method for the detection and quantitation of whole egg, whole milk, peanut, and hazelnut in eight food matrices was developed and evaluated in a single-laboratory validation. The matrices include cookies, cookie dough, bread, breakfast cereal, salad dressing, ice cream, and red wine.

Objective: The method was compared with Standard Method Performance Requirements (SMPR) 2016.

Simultaneous Analysis of Multiple Allergens in Food Products by LC-MS/MS.

There is currently no cure for food allergies, and sufferers can only rely on the correct labeling of foods to avoid allergens. Hence, it is important that analytical methods are sensitive and accurate enough to screen for the presence of multiple allergens in food products. In this study, we developed an LC-tandem MS method that is able to simultaneously screen or quantify the signature tryptic peptides of multiple allergen commodities.

Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model.

The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC, we performed a near-saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers and a very large number (2,860) of candidate later stage drivers that were enriched for genes that are mutated, deregulated or functioning in signaling pathways important for human HCC, with a striking 1,199 genes being linked to cellular metabolic processes.

Optimizing hollow-fiber-based pharmacokinetic assay via chemical stability study to account for inaccurate simulated drug clearance of rifampicin.

With increasing multidrug resistance coupled to a poor development pipeline, clinicians are exploring antimicrobial combinations to improve treatment outcomes. In vitro hollow-fiber infection model (HFIM) is employed to simulate human in vivo drug clearance and investigate pharmacodynamic synergism of antibiotics. Our overarching aim was to optimize the HFIM-based pharmacokinetic (PK) assay by using rifampicin and polymyxin B as probe drugs.

Pyrrolidinediones reduce the toxicity of thiazolidinediones and modify their anti-diabetic and anti-cancer properties.

Thiazolidinediones have been established as a drug class of significant importance in the treatment of Type II diabetes mellitus and have more recently displayed emergent potential as anti-cancer agents. However, their toxicity has hampered clinical development and usage in both therapeutic areas. Studies to date have implicated that the thiazolidinedione ring is responsible for the generation of reactive metabolites after metabolism.

Interaction of lapatinib with cytochrome P450 3A5.

Lapatinib, an oral tyrosine kinase inhibitor used for breast cancer, has been reported to cause idiosyncratic hepatotoxicity. Recently, it has been found that lapatinib forms a metabolite-inhibitor complex (MIC) with CYP3A4 via the formation of an alkylnitroso intermediate. Because CYP3A5 is highly polymorphic compared with CYP3A4 and also oxidizes lapatinib, we investigated the interactions of lapatinib with CYP3A5.

Preclinical metabolism and disposition of SB939 (Pracinostat), an orally active histone deacetylase inhibitor, and prediction of human pharmacokinetics.

The preclinical absorption, distribution, metabolism, and excretion (ADME) properties of Pracinostat [(2E)-3-[2-butyl-1-[2-(diethylamino) ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide hydrochloride; SB939], an orally active histone deacetylase inhibitor, were characterized and its human pharmacokinetics (PK) was predicted using Simcyp and allometric scaling. SB939 showed high aqueous solubility with high Caco-2 permeability. Metabolic stability was relatively higher in dog and human liver microsomes than in mouse and rat.

Reversible time-dependent inhibition of cytochrome P450 enzymes by duloxetine and inertness of its thiophene ring towards bioactivation.

Duloxetine is a selective serotonin-norepinephrine reuptake inhibitor (SNRI) approved to treat major depressive disorder and diabetic peripheral neuropathic pain. It is known to cause hepatotoxicity, in some cases leading to death. It has been reported that duloxetine causes time-dependent inhibition (TDI) of CYP1A2, CYP2B6, CYP2C19 and CYP3A4/5; but the nature of these TDI (whether reversible or irreversible) is not known.

Metabolic intermediate complex formation of human cytochrome P450 3A4 by lapatinib.

Lapatinib, an oral breast cancer drug, has recently been reported to be a mechanism-based inactivator of cytochrome P450 (P450) 3A4 and also an idiosyncratic hepatotoxicant. It was suggested that formation of a reactive quinoneimine metabolite was involved in mechanism-based inactivation (MBI) and/or hepatotoxicity. We investigated the mechanism of MBI of P450 3A4 by lapatinib.

Metabolic profiling of 3-nitropropionic acid early-stage Huntington's disease rat model using gas chromatography time-of-flight mass spectrometry.

3-Nitropropionic acid (3-NP), a potent irreversible inhibitor of mitochondrial complex II enzyme, leads to mitochondrial dysfunction and oxidative stress in Huntington's disease (HD) rat model. In this study, biochemical assays were used to demonstrate the presence of oxidative stress and mitochondrial dysfunction in 3-NP early stage HD rat models. Gas chromatography time-of-flight mass spectrometry (GC/TOFMS) was applied to analyze metabolites in brain and plasma of 3-NP-treated and vehicle-dosed rats.

Mechanism-based inactivation of cytochrome P450 3A4 by lapatinib.

Fatalities stemming from hepatotoxicity associated with the clinical use of lapatinib (Tykerb), an oral dual tyrosine kinase inhibitor (ErbB-1 and ErbB-2) used in the treatment of metastatic breast cancer, have been reported. We investigated the inhibition of CYP3A4 by lapatinib as a possible cause of its idiosyncratic toxicity. Inhibition of CYP3A4 was time-, concentration-, and NADPH-dependent, with k(inact) = 0.

Direct toxicity effects of sulfo-conjugated troglitazone on human hepatocytes.

Troglitazone (TGZ), an orally active hypoglycemic agent, was found to be associated with severe drug-induced liver failure and was withdrawn from the market in 2000. Although the exact mechanism is not clear, it has been postulated that the formation of its major sulfo-conjugated metabolite (TGZS) plays an important role in its toxicity. TGZS inhibits bile salt export pump (BSEP) that causes accumulation of bile salts in liver.

Ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) for time-dependent profiling of raw and steamed Panax notoginseng.

The metabolic profiles of Panax notoginseng and its associated therapeutic values are critically affected by the duration of steaming. The time-dependent steaming effect of P. notoginseng is not well-characterized and there is also no official guideline on its duration of steaming.

Investigation of the role of the thiazolidinedione ring of troglitazone in inducing hepatotoxicity.

Troglitazone (TGZ) is an orally active hypoglycemic agent which is used for the treatment of non-insulin-dependent diabetes mellitus. It had been associated with severe drug-induced liver failure which resulted in its withdrawal from the market in 2000. While the exact mechanism of its toxicity remains unknown, it has been postulated that the formation of toxic reactive metabolites (RMs) may play an important role in the hepatotoxicity of TGZ.

Metabolic profiling of rat brain and cognitive behavioral tasks: potential complementary strategies in preclinical cognition enhancement research.

In this study, the correlation between the metabolic profiles of rats undergoing cognition enhancement drug therapy and their associated cognitive behavioral outcomes were investigated. Male Lister Hooded rats were administered either with donepezil, galantamine, or vehicle and subjected to Atlantis watermaze training and novel object recognition tests. An UPLC/MS/MS method was developed to profile 21 neurologically related metabolites in the rat brains.

A sensitive LC/MS/MS bioanalysis assay of orally administered lipoic acid in rat blood and brain tissue.

A sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) bioanalytical method was developed and validated to analyze lipoic acid (LA) in rat blood and brain samples. Ten mobile phase combinations were investigated during method development. Mobile phase combination of 0.

Oxidative bioactivation and toxicity of leflunomide in immortalized human hepatocytes and kinetics of the non-enzymatic conversion to its major metabolite, A77 1726.

We used immortalized human hepatocytes to study the bioactivation of leflunomide and the metabolic degradation to its major metabolite, A77 1726. Both leflunomide and A77 1726 caused a time- and concentration-dependent increase in LDH release. The cytotoxicity of leflunomide, but not that of A77 1726, was prevented by the pan-CYP inhibitor, 1-aminobenzotriazole, indicating that an oxidative metabolite(s) was responsible for the cell injury.

Pharmaceutical metabolite profiling using quadrupole/ion mobility spectrometry/time-of-flight mass spectrometry.

The use of hybrid quadrupole ion mobility spectrometry time-of-flight mass spectrometry (Q/IMS/TOFMS) in the metabolite profiling of leflunomide (LEF) and acetaminophen (APAP) is presented. The IMS drift times (T(d)) of the drugs and their metabolites were determined in the IMS/TOFMS experiments and correlated with their exact monoisotopic masses and other in silico generated structural properties, such as connolly molecular area (CMA), connolly solvent-excluded volume (CSEV), principal moments of inertia along the X, Y and Z Cartesian coordinates (MI-X, MI-Y and MI-Z), inverse mobility and collision cross-section (CCS). The correlation of T(d) with these parameters is presented and discussed.

Investigation of the drug-drug interaction between alpha-lipoic acid and valproate via mitochondrial beta-oxidation.

Purpose: To investigate the potential drug-drug interaction (DDI) between lipoic acid (LA) and valproate (VA) via the mitochondrial beta-oxidation pathway in rats.

Methods: In vitro mitochondrial assays were performed to compare the biotransformation of VA to valproyl-CoA (VA-CoA), in the absence and presence of LA. In vitro microsomal and protein binding assays were performed to elucidate their potential DDI at the microsomal metabolism and distribution levels.

Prevention of acetaminophen (APAP)-induced hepatotoxicity by leflunomide via inhibition of APAP biotransformation to N-acetyl-p-benzoquinone imine.

Acetaminophen (APAP) is safe at therapeutic levels but causes liver injury via N-acetyl-p-benzoquinone imine (NAPQI)-induced oxidative stress when overdose. Recent studies indicated that mitochondrial permeability transition (mPT) plays a key role in APAP-induced toxicity and leflunomide (LEF) protects against the toxicity through inhibition of c-jun NH2-terminal protein kinase (JNK)-mediated pathway of mPT. It is not clearly understood if LEF also exerts its protective effect through inhibition of APAP bioactivation to the toxic NAPQI.

Evaluation of BEH C18, BEH HILIC, and HSS T3 (C18) column chemistries for the UPLC-MS-MS analysis of glutathione, glutathione disulfide, and ophthalmic acid in mouse liver and human plasma.

Glutathione (GSH), glutathione disulfide (GSSG), and ophthalmic acid (OA) are important biological oxidative stress biomarkers to be monitored in pathological and toxicological studies. With the advent of liquid chromatography tandem mass spectrometry (LC-MS-MS) technology, sensitive and selective analysis of these biomarkers in biological samples is now being performed routinely. Due to the hydrophilic and polar natures of GSH and its endogenous derivatives, achieving good retention, resolution, and peak shape is often a chromatographic challenge.

In vitro metabolism of leflunomide by mouse and human liver microsomes.

Leflunomide was found to be metabolized predominantly to A77-1726 and two novel hydroxylated metabolites, M1 and M2, in microsomes while A77-1726 was only biotransformed to M1. M1 and M2 were proposed to be the hydroxylated alpha-cyanoenol form of A77-1726 and the hydroxylated 5 methyl-isoxazole form of leflunomide, respectively.

In vitro phase I cytochrome P450 metabolism, permeability and pharmacokinetics of SB639, a novel histone deacetylase inhibitor in preclinical species.

In vitro liver microsomal stability, permeability, pharmacokinetics (PK) and oral bioavailability of SB639, a novel HDACi (Histone Deacetylase inhibitor), were determined. The in vitro metabolism was examined in mouse, rat, dog and human liver microsomes. The permeability and efflux potential of SB639 were determined using Caco-2 cell monolayers.

Pharmacokinetic study of intraperitoneally administered troglitazone in mice using ultra-performance liquid chromatography/tandem mass spectrometry.

A rapid and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the determination of troglitazone in mouse plasma. Troglitazone and its internal standard (IS), rosiglitazone, were separated on an ACQUITY UPLC BEH C(18) column (1.7 microm particle size, 50 x 2.

Development and validation of high-performance liquid chromatography-tandem mass spectrometry assay for 6-(3-benzoyl-ureido)-hexanoic acid hydroxyamide, a novel HDAC inhibitor, in mouse plasma for pharmacokinetic studies.

A liquid chromatography/tandem mass spectrometric method for the quantification of 6-(3-benzoyl-ureido)-hexanoic acid hydroxyamide (EX-2), a novel histone deacetylase (HDAC) inhibitor, in mouse plasma was developed to support in-house pharmacokinetic (PK) studies in the lead optimization stage. In order to determine the PK parameters for EX-2 in comparison to other HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA), PXD-101 and LBH-589, which are currently in different stages of clinical trials, research-grade bio-analytical method validations were carried out for EX-2 and these reference HDAC inhibitors, which were synthesized by in-house medicinal chemists. The components of validation consisted of specificity, extraction efficiency, signal-response of calibration standards, lower limit of quantification, autosampler stability and accuracy and precision of quality control samples.