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. An ultrapressure liquid chromatography tandem mass spectrometry method was validated for the quantification of rifampicin and polymyxin B components. In vitro profiling studies demonstrated that the experimental PK profiles of polymyxin B monotherapy were well correlated with the human population PK data while monotherapy with rifampicin failed to achieve the expected maximum plasma concentration. Chemical stability studies confirmed polymyxin B was stable in broth at 37 °C up to 12 h while rifampicin was unstable under the same conditions over 12 and 80 h. The calculated mean clearance of rifampicin due to chemical degradation was 0.098 ml/min accounting for 12.2 % of its clinical total clearance (CL = 0.8 ml/min) based on population PK data. Our novel finding reinforces the importance to optimize HFIM-based PK assay by performing chemical stability study so as to account for potential discrepancy between experimental and population PK profiles of antimicrobial agents.

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. Lapatinib inactivated CYP3A5 in a time-, concentration-, and NADPH-dependent manner using testosterone as a probe substrate with K(I) and k(inact) values of 0.0376 mM and 0.0226 min(-1), respectively. However, similar results were not obtained when midazolam was used as the probe substrate, suggesting that inactivation of CYP3A5 by lapatinib is site-specific. Poor recovery of CYP3A5 activity postdialysis and the lack of a Soret peak confirmed that lapatinib does not form a MIC with CYP3A5. The reduced CO difference spectrum further suggested that a large fraction of the reactive metabolite of lapatinib is covalently adducted to the apoprotein of CYP3A5. GSH trapping of a reactive metabolite of lapatinib formed by CYP3A5 confirmed the formation of a quinoneimine-GSH adduct derived from the O-dealkylated metabolite of lapatinib. In silico docking studies supported the preferential formation of an O-dealkylated metabolite of lapatinib by CYP3A5 compared with an N-hydroxylation reaction that is predominantly catalyzed by CYP3A4. In conclusion, lapatinib appears to be a mechanism-based inactivator of CYP3A5 via adduction of a quinoneimine metabolite.

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. The orthogonal partial least-squares discriminant analysis (OPLS-DA) model generated using brain metabolic profiles robustly differentiated the 3-NP early stage HD rat model from the control. Metabonomic characterization of the 3-NP HD rat model facilitated the detection of biomarkers that define the physiopathological phenotype of early stage HD and elucidated the treatment effect of galantamine. Brain marker metabolites that were identified based on the OPLS-DA model were associated with altered glutathione metabolism, oxidative stress, and impaired energy metabolism. The treatment effect of galantamine in early stage HD could not be concluded mechanistically using the brain metabotype. Our study confirmed that GC/TOFMS is a strategic and complementary platform for the metabonomic characterization of 3-NP induced neurotoxicity in the early stage HD rat model.

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. Liquid chromatography-mass spectrometry analysis of P450 3A4 after incubation with lapatinib did not show any peak corresponding to irreversible modifications. The enzymatic activity inactivated by lapatinib was completely restored by the addition of potassium ferricyanide. These results indicate that the mechanism of MBI by lapatinib is quasi-irreversible and mediated via metabolic intermediate complex (MI complex) formation. This finding was verified by the increase in a signature Soret absorbance at approximately 455 nm. Two amine oxidation products of the metabolism of lapatinib by P450 3A4 were characterized: N-hydroxy lapatinib (M3) and the oxime form of N-dealkylated lapatinib (M2), suggesting that a nitroso or another related intermediate generated from M3 is involved in MI complex formation. In contrast, P450 3A5 was much less susceptible to MBI by lapatinib via MI complex formation than P450 3A4. In addition, P450 3A5 had a significantly lower ability than 3A4 to generate M3, consistent with N-hydroxylation as the initial step in the pathway to MI complex formation. In conclusion, our results demonstrate that the primary mechanism for MBI of P450 3A4 by lapatinib is not irreversible modification by the quinoneimine metabolite, but quasi-irreversible MI complex formation mediated via oxidation of the secondary amine group of lapatinib.

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. The major metabolites formed in human liver microsomes were also observed in preclinical species. Human cytochrome P450 (P450) phenotyping showed that SB939 was primarily metabolized by CYP3A4 and CYP1A2. SB939 did not significantly inhibit human CYP3A4, 1A2, 2D6, and 2C9 (>25 µM) but inhibited 2C19 (IC(50) = 5.8 µM). No significant induction of human CYP3A4 and 1A2 was observed in hepatocytes. Plasma protein binding in mouse, rat, dog, and human ranged between ∼84 and 94%. The blood-to-plasma ratio was ∼1.0 in human blood. SB939 showed high systemic clearance (relative to liver blood flow) of 9.2, 4.5, and 1.5 l · h(-1) · kg(-1) and high volume of distribution at steady state (>0.6 l/kg) of 3.5, 1.7, and 4.2 l/kg in mouse, rat, and dog, respectively. The oral bioavailability was 34, 65, and ∼3% in mice, dogs, and rats, respectively. The predicted oral PK profile and parameters of SB939, using Simcyp and allometric scaling, were in good agreement with observed data in humans. Simcyp predictions showed lack of CYP3A4 and 2C19 drug-drug interaction potential for SB939. In summary, the preclinical ADME of SB939 supported its preclinical and clinical development as an oral drug candidate.

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) = 0.0202 min(-1) and K(i) = 1.709 µM. The partition ratio was approximately 50.9. Addition of GSH did not affect the rate of inactivation. Testosterone protected CYP3A4 from inactivation by lapatinib. The characteristic Soret peak associated with a metabolite-intermediate complex was not observed for lapatinib during spectral difference scanning. However, reduced carbon monoxide (CO)-difference spectroscopy did reveal a 43% loss of the spectrally detectable CYP3A4-CO complex in the presence of lapatinib. Incubation of either lapatinib or its dealkylated metabolite with human liver microsomes in the presence of GSH resulted in the formation of a reactive metabolite (RM)-GSH adduct derived from the O-dealkylated metabolite of lapatinib. In addition, coincubation of lapatinib with ketoconazole inhibited the formation of the RM-GSH adduct. In conclusion, we demonstrated for the first time that lapatinib is a mechanism-based inactivator of CYP3A4, most likely via the formation and further oxidation of its O-dealkylated metabolite to a quinoneimine that covalently modifies the CYP3A4 apoprotein and/or heme moiety.

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) 2016.002 established by the AOAC Stakeholder Panel on Strategic Food Analytical Methods. METHODS: The method involves tryptic digestion of allergen proteins in food matrices incurred or spiked with allergen standards [reference materials (RMs), Standard RMs (SRMs), or in-house prepared standard] and uses labeled peptide internal standards. LC-tandem MS analysis of the signature tryptic peptides of the four allergens is performed using multiple reaction monitoring. RESULTS: For 10 allergen/matrix combinations, the method demonstrated adequate sensitivity with a minimum quantitation limit of 3 mg/kg for whole egg and 10 mg/kg for milk, peanut, and hazelnut allergens. Repeatability precision across 3 days of analyses was <17% with analytical range of 10-1000 mg/kg. Recovery from incurred and spiked matrix-matched standards varied from 60 to 118%. CONCLUSIONS: The method met the minimum performance requirements of SMPR 2016.002 for whole egg in cookies, bread, cookie dough, and salad dressing; whole milk in cookies and red wine; peanut in breakfast cereal; and hazelnut in cookies. HIGHLIGHTS: The ERP determined that the data presented met the SMPR and accordingly recommended the method to be granted First Action status. In September 2017, the Official Methods Board approved the method as First Action.

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. Pharmacologically induced behavioral changes were compared subsequently with the metabolic fluctuations of neurologically related metabolites from multiple neurotransmitter pathways using multivariate and univariate statistical analyses. Significant improvements in cognitive behavioral outcomes were demonstrated in the rats administered with donepezil and galantamine using both AWM training (P < 0.05) and NOR (P < 0.05) tests as compared to those dosed with the vehicle. This corroborated with the significant elevation of eight prominent biomarkers after the cognitive enhancement therapy. An orthogonal partial least-squares discriminant analysis model generated using only the 8 metabolites identified as discriminating the drug-dosed rats from the vehicle-dosed rats gave a Q(2) = 0.566, receiver operator characteristic (ROC) AUC = 1.000, using 7-fold cross validation. Our study suggests that metabolic profiling of rat brain is a potential complementary strategy to the cognitive behavioral tasks for characterizing neurobiological responses to cognition enhancement drug testing.

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. The present work was undertaken to study the effect of LEF on the bioactivation of APAP to NAPQI. Mechanism-based inhibition incubations performed in mouse and human liver microsomes (MLM and HLM) indicated that inhibition of APAP bioactivation to NAPQI was observed in MLM but not in HLM. Furthermore, LEF but not its active metabolite, A77-1726, was shown to be the main inhibitor. When APAP and LEF were incubated with human recombinant P450 enzymes, CYP1A2 was found to be the isozyme responsible for the inhibition of APAP bioactivation. Species variation in CYP1A2 enzymes probably accounted for the different observations in our MLM and HLM studies. We concluded that inhibition of NAPQI formation is not a probable pathway that LEF protects APAP-induced hepatotoxicity in human.

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 this study, three ultra-performance (UP) LC column chemistries (namely, BEH C18, BEH HILIC, and HSS T3 [C18]) are evaluated for the UPLC-MS-MS analysis of GSH, GSSG, and OA extracted from mouse liver and human plasma samples. The chromatographic parameters evaluated are retentivity, tailing factor, MS sensitivity, and resolution of the three analytes. Based on the optimized method for each column chemistry, our results indicate that the HSS T3 (C18) column chemistry affords the best retention and separation of these analytes when operated under the ultra high-pressure chromatographic conditions.

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. This method is capable of screening and identifying egg white, skim milk, peanut, soy, and tree nuts (almond, Brazil nut, cashew, hazelnut, pecan, pine nut, pistachio, and walnut) at a detection limit of 10 ppm in incurred bread and cookies. It was further tested for the quantitative analysis of whole-egg, whole-milk, peanut butter, and hazelnut commodities, which are incurred or spiked into selected food matrixes as defined in AOAC INTERNATIONAL Standard Method Performance Requirement (SMPR®) 2016.002. The method demonstrated excellent sensitivity with a Method quantitative limit of 3 ppm for whole egg and 10 ppm for the remaining three allergen commodities. It also demonstrated good recovery (60-119%) and repeatability (RSDr <20%), with an analytical range of 10-1000 ppm for each allergen commodity and was able to meet the minimum performance requirements of the SMPR.

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. Our study provides a comprehensive overview of the genetic landscape of HCC.

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. As an attempt to improve their safety profiles, we considered the bioisosteric replacement of the thiazolidinedione ring with a chemically conserved pyrrolidinedione heterocyclic system. Using pyrrolidinedione analogs of the thiazolidinedione drugs troglitazone (TGZ), rosiglitazone (RGZ), and pioglitazone (PGZ), we evaluated their PPAR(γ) activities, anti-cancer properties as well as toxicological effects. Of significance, both pyrrolidinedione analogs demonstrated reduced toxicity. Pharmacologically, they also displayed diminished PPAR(γ) binding and ap2 gene expression in a mouse pre-adipocyte cell line 3T3-L1, but enhanced anti-cancer properties based on the suppression of liver cancer cell line (Huh-7) proliferation and the expression of tumor marker, afp. Overall, this study ascertains the general contribution of the thiazolidinedione ring to their cytotoxicity and proposes the applicability of the pyrrolidinedione ring as a selective and safer choice in anti-diabetic and cancer chemotherapeutics for future drug design.

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. Irreversible TDI can cause clinically significant drug-drug interactions and also immune-mediated hepatotoxicity. Structurally, duloxetine possesses several toxicophores, i.e. the naphthyl and thiophene rings. It has been reported that the naphthyl ring undergoes epoxidation and was subsequently adducted to glutathione, but bioactivation related to the thiophene ring has not been completely elucidated. In this paper, the potential of duloxetine in causing irreversible TDI and generating reactive metabolites was investigated. Human liver microsomal assays demonstrated that duloxetine did not cause irreversible TDI of CYP1A2, CYP2B6, CYP2D6, CYP2C19 and CYP3A4/5. Subsequently, reactive metabolite trapping assays using soft nucleophiles (glutathione and glutathione ethyl ester) revealed a previously reported adduct at the naphthyl ring of duloxetine but not at the thiophene ring. Trapping assays utilizing a hard nucleophile (semicarbazide) did not demonstrate adducts with the thiophene ring, indicating an absence of thiophene ring opening. The hepatotoxicity of duloxetine is possibly not related to the irreversible TDI of CYP450 or the bioactivation of its thiophene moiety, but might be due to the epoxidation of its naphthyl ring.

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. The purpose of this study is to investigate the role of sulfur moiety of thiazolidinedione (TZD) nucleus in inducing liver toxicity via the formation of RMs. An analogue of TGZ, trosuccinimide (TSN), was synthesized chemically where the sulfur moiety of thiazolidinedione ring was replaced by a methylene group. Both compounds were incubated independently with human liver microsomes enriched with glutathione (GSH) and normal human hepatocytes (THLE-2 cell lines) to profile GSH-adducts using ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS). Four RM-GSH conjugates of TGZ were identified during the profiling experiments of which three were related to the sulfur moiety of the TZD ring, whereas no RM of TSN was detected in both microsomes and hepatocytes. MTT, GSH and protein carbonyl (PC) assays were performed using THLE-2 hepatocytes to measure the levels of toxicity of TGZ and TSN in vitro. Finally, peroxisome proliferator activated receptor gamma (PPAR(gamma)) binding activity was measured to check the binding affinities of both TGZ and TSN. The calculated binding affinities of TGZ and TSN were 332.2 and 1106.0 microM, respectively. Our results indicated collectively that TSN (EC(50)=138.5+/-7.32 microM) was less toxic than TGZ (EC(50)=27.2+/-4.8 microM) in THLE-2 hepatocytes. As both compounds were shown to bind to PPAR(gamma), the substitution of the TZD moiety may be beneficial from a drug design perspective. In conclusion, our study confirmed that the TZD ring of TGZ may be partially responsible for its liver toxicity in humans via the formation of RMs.

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. In this paper, a UHPLC/TOFMS-based metabolomic platform was developed for the qualitative profiling of multiparametric metabolic changes of raw P. notoginseng during the steaming process. Our method was successful in discriminating the differentially processed herbs. Both the unsupervised principal component analysis (PCA) score plot (R(2)X=0.664, Q(2) (cum)=0.622, and PCs=2) and the supervised partial least square-data analysis (PLS-DA) model (R(2)X=0.708, R(2)Y=0.461, and Q(2)Y=0.271) demonstrated strong classification and clear trajectory patterns with regard to the duration of steaming. The PLS-DA model was validated for its robustness via a prediction set, confirming that the UHPLC/TOFMS metabolic profiles of the raw and differentially steamed P. notoginseng samples were highly reproducible. Based on our method, the minimum durations of steaming for the maximum production of bioactive ginsenosides such as Rg3 and Rh2 were also predicted. Our novel time-dependent metabolic profiling approach represents the paradigm shift in the quality control of P. notoginseng products.

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. High concentration of bile salts causes cell death and mitochondrial dysfunction via detergent properties. One question arises whether TGZS has direct toxicity effect on human liver cells in addition to BSEP inhibition. In this study, both TGZ and chemically synthesized TGZS were incubated with normal human hepatocytes (THLE-2 cells) for measuring their cytotoxicity in vitro using the MTT assay. Glutathione (GSH) and protein carbonyl (PC) assays were further performed to measure the oxidative stress generated by these two compounds during incubation with THLE-2 cells. The results from this study indicated that TGZS (EC(50)=21.74+/-5.38 microM) was more toxic than TGZ (EC(50)=41.12+/-4.3 microM) in THLE-2 cells. The GSH and PC data further confirmed that TGZS produced greater oxidative stress in THLE-2 cells as compared to TGZ. In conclusion, our study demonstrated for the first time that TGZS has direct toxicity effect on human liver cells and may be partially responsible for the hepatotoxicity of TGZ.

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 0.1% acetic acid (pH 4 adjusted with ammonia solution)/acetonitrile was most optimum in terms of sensitivity and peak shape of LA and the internal standard, valproic acid. Sample extraction method was explored using liquid-liquid extraction and protein precipitation methods. Protein precipitation yielded the highest recovery of the analytes from blood and brain ranging from 92 to 115%. The lower limit of quantitation (LLOQ) of LA was 0.1ng/mL (0.485nM) in both blood and brain while on-column lower limit of detection (LLOD) was 0.03pg. The precision (% R.S.D.) ranged from 1.49 to 26.39% and 1.49 to 10.89% for intra- and inter-day assays, respectively. The accuracy ranged from 91.2 to 116.17% for intra-day assay and 102.68 to 114.33% for inter-day assay.

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. IMS/TOF tandem mass spectrometry experiments (MS(2) and MS(3)) were successfully performed on the N-acetyl-p-benzoquinoneimine glutathione (NAPQI-GSH) adduct derived from the in vitro microsomal metabolism of APAP. As comparison, similar experiments were also performed using hybrid triple quadrupole linear ion trap mass spectrometry (QTRAPMS) and quadrupole time-of-flight mass spectrometry (QTOFMS). The abilities to resolve the product ions of the metabolite within the drift tube and fragment the ion mobility resolved product ions in the transfer travelling wave-enabled stacked ring ion guide (TWIG) demonstrated the potential applicability of the Q/IMS/TOFMS technique in pharmaceutical metabolite profiling.

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. LC/MS/MS analysis revealed that leflunomide was rapidly degraded in hepatocytes biphasically (t((1/2))(a) = 1.5 h, t((1/2)) >24 h), but much slower in cell-free medium (t((1/2)) >24 h). In contrast, the generation of A77 1726 occurred at a similar rate in cells and cell-free systems. In conclusion, leflunomide was rapidly metabolized in human hepatocytes to A77 1726, but its toxicity was dependent on other, CYP-dependent intermediates.