Surrogate matrix and surrogate analyte approaches for definitive quantitation of endogenous biomolecules.

BACKGROUND: Quantitation of biomarkers by LC-MS/MS is complicated by the presence of endogenous analytes. This challenge is most commonly overcome by calibration using an authentic standard spiked into a surrogate matrix devoid of the target analyte. A second approach involves use of a stable-isotope-labeled standard as a surrogate analyte to allow calibration in the actual biological matrix. For both methods, parallelism between calibration standards and the target analyte in biological matrix must be demonstrated in order to ensure accurate quantitation. RESULTS: In this communication, the surrogate matrix and surrogate analyte approaches are compared for the analysis of five amino acids in human plasma: alanine, valine, methionine, leucine and isoleucine. In addition, methodology based on standard addition is introduced, which enables a robust examination of parallelism in both surrogate analyte and surrogate matrix methods prior to formal validation. Results from additional assays are presented to introduce the standard-addition methodology and to highlight the strengths and weaknesses of each approach. CONCLUSION: For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.

N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a novel metabotropic glutamate 2 potentiator with potential anxiolytic/antidepressant properties: in vivo profiling suggests a link between behavioral and central nervous system neurochemical changes.

The normalization of excessive glutamatergic neurotransmission through the activation of metabotropic glutamate 2 (mGlu2) receptors may have therapeutic potential in a variety of psychiatric disorders, including anxiety/depression and schizophrenia. Here, we characterize the pharmacological properties of N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a structurally novel, potent, and selective allosteric potentiator of human and rat mGlu2 receptors (EC(50) = 23 and 13 nM, respectively). THIIC produced anxiolytic-like efficacy in the rat stress-induced hyperthermia assay and the mouse stress-induced elevation of cerebellar cGMP and marble-burying assays. THIIC also produced robust activity in three assays that detect antidepressant-like activity, including the mouse forced-swim test, the rat differential reinforcement of low rate 72-s assay, and the rat dominant-submissive test, with a maximal response similar to that of imipramine. Effects of THIIC in the forced-swim test and marble burying were deleted in mGlu2 receptor null mice. Analysis of sleep electroencephalogram (EEG) showed that THIIC had a sleep-promoting profile with increased non-rapid eye movement (REM) and decreased REM sleep. THIIC also decreased the dark phase increase in extracellular histamine in the medial prefrontal cortex and decreased levels of the histamine metabolite tele-methylhistamine (t-MeHA) in rat cerebrospinal fluid. Collectively, these results indicate that the novel mGlu2-positive allosteric modulator THIIC has robust activity in models used to predict anxiolytic/antidepressant efficacy, substantiating, at least with this molecule, differentiation in the biological impact of mGlu2 potentiation versus mGlu2/3 orthosteric agonism. In addition, we provide evidence that sleep EEG and CSF t-MeHA might function as viable biomarker approaches to facilitate the translational development of THIIC and other mGlu2 potentiators.

Analysis of glutamine, glutamate, pyroglutamate, and GABA in cerebrospinal fluid using ion pairing HPLC with positive electrospray LC/MS/MS.

A simple and sensitive method for the separation and quantitation of glutamine, glutamate, pyroglutamate, and gamma-aminobutyric acid (GABA) in cerebrospinal fluid (CSF) is presented. The method utilizes ion pairing with heptafluorobutyric acid (HFBA) to achieve HPLC separation with detection by positive ESI LC/MS/MS. The method does not require extraction or derivatization, utilizes a heavy labeled internal standard for each analyte, and allows for rapid throughput with a 5 min run time. The method was developed with particular attention taken to prevent conversion between analytes known to occur under certain conditions. The lower limit of quantitation is 7.8 ng/ml for all analytes, and the intra-day and inter-day accuracy (%RE) and precision (%R.S.D.) are defined for all analytes. The method was developed as a sensitive, selective, and robust method to investigate the excitatory and inhibitory neurotransmitters (glutamate and GABA) as biomarkers in drug development.

Simultaneous profiling of multiple neurochemical pathways from a single cerebrospinal fluid sample using GC/MS/MS with electron capture detection.

Biogenic amines and amino acids are widely characterized in the pathways representing neurotransmission. Although several analytical methodologies have been used to detect specific target molecules in relevant fluids such as cerebrospinal fluid (CSF), multiple assays must be used to survey the primary pathways involved. This article describes the development of a GC/MS/MS method capable of analyzing up to 43 analytes (representing 20 amino acids and more than seven neurochemical pathways) from a single 50 microl CSF sample. In this procedure, a CSF sample is first treated with acetonitrile to precipitate proteins. The dried sample is then derivatized with a mixture of 2,2,3,3,3-pentafluoro-1-propanol and pentafluoropropionic acetic anhydride to replace all active hydrogen atoms with fluorine-containing groups. Due to the concentration difference between amino acids and neurotransmitters, these two compound classes are analyzed in separate injections of the same derivatized extract. The total run time for each injection is approximately 15-20 min. An essential feature of the method is the use of argon as a reagent gas for electron capture chemical ionization (ECCI), as the use of the more traditional gas (methane) lacked sufficient durability to be considered for use with the present instrumentation. This article describes the development of this method including a detailed investigation of the chemical ionization conditions used. The resultant conditions allow for the profiling of biogenic amines (e.g. serotonin, norepinephrine, and dopamine) in the low picogram per milliliter range.

Current applications of liquid chromatography/mass spectrometry in pharmaceutical discovery after a decade of innovation.

Current drug discovery involves a highly iterative process pertaining to three core disciplines: biology, chemistry, and drug disposition. For most pharmaceutical companies the path to a drug candidate comprises similar stages: target identification, biological screening, lead generation, lead optimization, and candidate selection. Over the past decade, the overall efficiency of drug discovery has been greatly improved by a single instrumental technique, liquid chromatography/mass spectrometry (LC/MS). Transformed by the commercial introduction of the atmospheric pressure ionization interface in the mid-1990s, LC/MS has expanded into almost every area of drug discovery. In many cases, drug discovery workflow has been changed owing to vastly improved efficiency. This review examines recent trends for these three core disciplines and presents seminal examples where LC/MS has altered the current approach to drug discovery.

Interactions of two major metabolites of prasugrel, a thienopyridine antiplatelet agent, with the cytochromes P450.

The biotransformation of prasugrel to R-138727 (2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid) involves rapid deesterification to R-95913 (2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone) followed by cytochrome P450 (P450)-mediated formation of R-138727, the metabolite responsible for platelet aggregation. For identification of the P450s responsible for the formation of the active metabolite, the current studies were conducted with R-95913 as the substrate. Incubations required supplementation with reduced glutathione. Hyperbolic kinetics (K(m) 21-30 microM), consistent with a single enzyme predominating, were observed after incubations with human liver microsomes. Correlation analyses revealed a strong relationship between R-138727 formation and CYP3A-mediated midazolam 1'-hydroxylation (r(2) = 0.98; p < 0.001) in a bank of characterized human liver microsomal samples. The human lymphoblast-expressed enzymes capable of forming R-138727, in rank order of rates, were CYP3A4>CYP2B6>CYP2C19 approximately CYP2C9>CYP2D6. A monoclonal antibody to CYP2B6 and the CYP3A inhibitor ketoconazole substantially inhibited R-138727 formation, whereas inhibitors of CYP2C9 (sulfaphenazole) and CYP2C19 (omeprazole) did not. Scaling of in vitro intrinsic clearance values from expressed enzymes to the whole liver using a relative abundance approach indicated that either CYP3A4 alone or CYP3A4 and CYP2B6 are the major contributors to R-138727 formation. R-95913 and R-138727 were also examined for their ability to inhibit metabolism mediated by five P450s. R-138727 did not inhibit the P450s tested. In vitro, R-95913 inhibited CYP2C9, CYP2C19, CYP2D6, and CYP3A, with K(i) values ranging from 7.2 microM to 82 microM, but did not inhibit CYP1A2. These K(i) values exceed circulating concentrations in humans by 3.8- to 43-fold. Therefore, neither R-95913 nor R-138727 is expected to substantially inhibit the P450-mediated metabolism of coadministered drugs.

An assessment of udp-glucuronosyltransferase induction using primary human hepatocytes.

Uridine diphosphate glucuronosyltransferases (UGTs) catalyze the glucuronidation of a wide range of xenobiotics and endogenous substrates. However, there is a lack of information concerning the response of human UGTs to inducers, and this observation prompted the current investigation. The glucuronidation of estradiol (3- and 17-positions), naphthol, propofol, and morphine (3- and 6-positions) was assessed against a battery of recombinant human UGTs to determine selective glucuronidation reactions for induction studies. The potential induction of the glucuronidation of estradiol at the 3-position, naphthol, propofol, and morphine at the 3-position was subsequently investigated in cultured primary human hepatocytes against a range of prototypic inducers including dexamethasone, 3-methylcholanthrene (3-MC), phenobarbital, rifampicin, and omeprazole. Treatment with 3-MC induced estradiol-3-glucuronidation (up to 2.5-fold) in four of five donors investigated. Statistically significant increases in naphthol glucuronidation (up to 1.7-fold) were observed following treatment with carbamazepine. UGT1A9-mediated propofol glucuronidation was induced by phenobarbital (up to 2.2-fold) and rifampicin (up to 1.7-fold). However, treatment with alpha-naphthoflavone and tangeretin resulted in a decrease in propofol glucuronidation (30% of control values). Statistically significant induction of morphine-3-glucuronidation was observed in at least three donors following treatment with phenobarbital, rifampicin, and carbamazepine. Each UGT isoform investigated displayed a distinct induction profile. Although statistically significant increases in glucuronidation were observed for each reaction studied, the level of induction was less than that observed for CYP1A2 or CYP3A4 and exhibited a large interdonor variability. The clinical relevance of the induction responses obtained in this study is unclear.

Identification of the human cytochromes P450 responsible for atomoxetine metabolism.

Studies were performed to determine the human enzymes responsible for the biotransformation of atomoxetine to its major metabolite, 4-hydroxyatomoxetine, and to a minor metabolite, N-desmethylatomoxetine. Utilizing human liver microsomes containing a full complement of cytochrome P450 (P450) enzymes, average K(m) and CL(int) values of 2.3 microM and 103 microl/min/mg, respectively, were obtained for 4-hydroxyatomoxetine formation. Microsomal samples deficient in CYP2D6 exhibited average apparent K(m) and CL(int) values of 149 microM and 0.2 microl/min/mg, respectively. In a human liver bank characterized for P450 content, formation of 4-hydroxyatomoxetine correlated only to CYP2D6 activity. Of nine expressed P450s examined, 4-hydroxyatomoxetine was formed at a rate 475-fold greater by CYP2D6 compared with the other P450s. These results demonstrate that CYP2D6 is the enzyme primarily responsible for the formation of 4-hydroxyatomoxetine. Multiple P450s were found to be capable of forming 4-hydroxyatomoxetine when CYP2D6 was not expressed. However, the efficiency at which these enzymes perform this biotransformation is reduced compared with CYP2D6. The formation of the minor metabolite N-desmethylatomoxetine exhibited average K(m) and CL(int) values of 83 microM and 0.8 microl/min/mg, respectively. Utilizing studies similar to those outlined above, CYP2C19 was identified as the primary enzyme responsible for the biotransformation of atomoxetine to N-desmethylatomoxetine. In summary, CYP2D6 was found to be the primary P450 responsible for the formation of the major oxidative metabolite of atomoxetine, 4-hydroxyatomoxetine. Furthermore, these studies indicate that in patients with compromised CYP2D6 activity, multiple low-affinity enzymes will participate in the formation of 4-hydroxyatomoxetine. Therefore, coadministration of P450 inhibitors to poor metabolizers of CYP2D6 substrates would not be predicted to decrease the clearance of atomoxetine in these individuals.