Determination of zanamivir in rat and monkey plasma by positive ion hydrophilic interaction chromatography (HILIC)/tandem mass spectrometry.

A hydrophilic interaction chromatography (HILIC)/mass spectrometric assay was developed for the determination of zanamivir, a neuraminidase inhibitor used to treat influenza, in rat and monkey plasma. An organic solvent with hydrophilic properties, methanol, was used to precipitate proteins in plasma to assure the highly polar zanamivir of staying in solution. Chromatographic separation was obtained using a HILIC silica column with multiple reaction monitoring turboionspray positive ion detection. The stable label of zanamivir, [(13)C(1)(15)N(2)] GR121167C, was used as the internal standard. The assay was validated for the determination of zanamivir in rat and monkey plasma. The lower and upper limits of quantitation were 2 and 10000 ng/mL, using 0.05 mL plasma aliquot, respectively. The signal to noise ratio of a typical 2 ng/mL was approximately 5:1. The inter-day precision (relative standard deviation) and accuracy (relative error) in rat plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 6 to 10% and -6.5 to 0.2%, respectively. The inter-day precision (relative standard deviation) and accuracy (relative error) in monkey plasma, derived from the analysis of validation samples at five concentrations, ranged from 2 to 8% and -2.3 to 2.1%, respectively. Zanamivir was found to be stable for at least 5 days at approximately -80 degrees C and at room temperature in plasma. This assay incorporates a simple protein precipitation with methanol and hydrophilic interaction chromatography which is sensitive, accurate, precise, and is being used to support oral formulation and toxicokinetic studies in rat and monkey, respectively.

Tryptophan-NAD+ pathway metabolites as putative biomarkers and predictors of peroxisome proliferation.

The present study was designed to provide further information about the relevance of raised urinary levels of N-methylnicotinamide (NMN), and/or its metabolites N-methyl-4-pyridone-3-carboxamide (4PY) and N-methyl-2-pyridone-3-carboxamide (2PY), to peroxisome proliferation by dosing rats with known peroxisome proliferator-activated receptor alpha (PPARalpha) ligands [fenofibrate, diethylhexylphthalate (DEHP) and long-chain fatty acids (LCFA)] and other compounds believed to modulate lipid metabolism via PPARalpha-independent mechanisms (simvastatin, hydrazine and chlorpromazine). Urinary NMN was correlated with standard markers of peroxisome proliferation and serum lipid parameters with the aim of establishing whether urinary NMN could be used as a biomarker for peroxisome proliferation in the rat. Data from this study were also used to validate a previously constructed multivariate statistical model of peroxisome proliferation (PP) in the rat. The predictive model, based on 1H nuclear magnetic resonance (NMR) spectroscopy of urine, uses spectral patterns of NMN, 4PY and other endogenous metabolites to predict hepatocellular peroxisome count. Each treatment induced pharmacological (serum lipid) effects characteristic of their class, but only fenofibrate, DEHP and simvastatin increased peroxisome number and raised urinary NMN, 2PY and 4PY, with simvastatin having only a transient effect on the latter. These compounds also reduced mRNA expression for aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase, EC 4.1.1.45), the enzyme believed to be involved in modulating the flux of tryptophan through this pathway, with decreasing order of potency, fenofibrate (-10.39-fold) >DEHP (-3.09-fold) >simvastatin (-1.84-fold). Of the other treatments, only LCFA influenced mRNA expression of ACMSDase (-3.62-fold reduction) and quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) (-2.42-fold) without any change in urinary NMN excretion. Although there were no correlations between urinary NMN concentration and serum lipid parameters, NMN did correlate with peroxisome count (r2=0.63) and acyl-CoA oxidase activity (r2=0.61). These correlations were biased by the large response to fenofibrate compared to the other treatments; nevertheless the data do indicate a relationship between the tryptophan-NAD+ pathway and PPARalpha-dependent pathways, making this metabolite a potentially useful biomarker to detect PP. In order to strengthen the observed link between the metabolites associated with the tryptophan-NAD+ pathway and more accurately predict PP, other urinary metabolites were included in a predictive statistical model. This statistical model was found to predict the observed PP in 26/27 instances using a pre-determined threshold of 2-fold mean control peroxisome count. The model also predicted a time-dependent increase in peroxisome count for the fenofibrate group, which is important when considering the use of such modelling to predict the onset and progression of PP prior to its observation in samples taken at autopsy.