In vitro interaction cocktail assay for nine major cytochrome P450 enzymes with 13 probe reactions and a single LC/MSMS run: analytical validation and testing with monoclonal anti-CYP antibodies

A sensitive and rugged LC/MSMS method was developed for a comprehensive in vitro metabolic interaction screening assay with N-in-1 approach reported earlier. A cocktail consisting of ten cytochrome P450 (CYP)-selective probe substrates with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8) tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were simultaneously analysed with a single LC/MSMS run. Altogether, 13 metabolites and internal standard phenacetin were analysed in multiple reaction mode. Polarity switching mode was utilized to acquire negative ion mode electrospray data for hydroxychlorzoxazone and positive ionization data for the rest of the analytes. Fast gradient elution was applied, giving total injection cycle of 8 min. The method was modified for two different LC/MSMS systems, and was validated for linear range, detection limit, accuracy and precision for each metabolite. In addition, cocktail inhibition system was further tested using monoclonal anti-CYP antibodies as inhibitors for each probe reaction.     

Proton radioactivity — spherical and deformed

The proton drip line defines one of the fundamental limits to nuclear stability. Nuclei lying beyond this line are energetically unbound to the emission of a constituent proton from their ground states. For near-spherical nuclei in the region of the drip line between Z=69 (Tm) and Z=81 (T1), proton decay transition rates have been shown to be well reproduced by WKB calculations using spectroscopic factors derived from a low-seniority shell model calculation [2]. Another approach using spectroscopic factors obtained from the independent quasiparticle approximation has also proved successful in this region [3]. These interpretations have allowed the extraction of nuclear structure information from nuclei well beyond the proton drip line.     

Fine Structure in the alpha decays of 226U and 230Pu

The nuclei ²²⁶U and ²³⁰Pu have been populated via reactions involving ²⁰⁸Pb targets bombarded by ²²Ne and ²⁶Mg projectiles. Fusion-evaporation residues were separated in-flight using a gas-filled recoil separator. A position-sensitive Si-strip detector was employed at the focal plane in order to identify correlated !-decay chains. Two fine structure !-decay lines have been observed. The first, with an energy of 7385(5) keV, is assigned as the ! decay from ²²⁶U to the first excited 2⁺ state of ²²²Th. The second line, observed for the first time in this work, has an energy of 6961(30) keV and is assigned as the ! decay from ²³⁰Pu to the first excited 2⁺ state of ²²⁶U. The excitation energy of the first excited 2⁺ state in ²²⁶U was determined to be 96(25) keV, giving an estimate of the deformation parameter #₂ ­ 0.20.     

Lifetime measurement in 195Po

The lifetime of the 17/2⁺ yrast state in ¹⁹⁵Po has been measured using the recoil distance Doppler-shift technique to be = 43(11) ps. The lifetime was extracted from the singles -ray spectra obtained by using the recoil-decay tagging method. The present work provides more information of the coupling schemes, shapes and configuration mixing in neutron-deficient odd-mass Po nuclei.     

Structure of rotational bands in 253No

In-beam gamma-ray and conversion electron spectroscopic studies have been performed on the ²⁵³ No nucleus. A strongly coupled rotational band has been identified and the improved statistics allows an assignment of the band structure as built on the $\ensuremath 9/2^-[734]_{\nu}$ ground state. The results agree with previously known transition energies but disagree with the tentative structural assignments made in earlier work.     

Spectroscopy and single-particle structure of the odd- Z heavy elements 255Lr, 251Md and 247Es

The odd-Z isotope ²⁵⁵Lr, its daughter ²⁵¹Md and grand-daughter ²⁴⁷Es were studied in two experiments performed at GANIL and the University of Jyv?skyl?. The ²⁵⁵Lr nuclei were produced using the cold fusion-evaporation reaction ²⁰⁹Bi(⁴⁸Ca, 2n)²⁵⁵Lr at a bombarding energy of 217MeV. The single-particle structure and decay properties were investigated using α, γ and electron spectroscopy. The ground-state spin and parity could be assigned for ²⁵⁵Lr (1/2^-) and ²⁵¹Md (7/2^-). States corresponding to the 7/2⁺[633], 7/2^-[514] and 1/2^-[521] Nilsson orbitals were observed. Results are compared to experimental data obtained in neighbouring isotopes and to Hartree-Fock-Bogoliubov calculations using the Skyrme interaction SLy4 and a density-dependent pairing interaction. The position of the 1/2^-[521] orbital from the spherical 2f _5/2 shell is discussed.     

Comparison of product ion spectra obtained by liquid chromatography/triple-quadrupole mass spectrometry for library search

Reproducibility of product ion spectra acquired using a liquid chromatography/triple-quadrupole mass spectrometry (LC/MS/MS) instrument over a 4-year period, and with three other LC/MS/MS instruments, one from the same manufacturer and two from a different manufacturer, was examined. The MS/MS spectra of 30 drug substances were generated in positive electrospray ionization mode at low, medium, and high collision energies (20, 35, and 50 eV). Purity and Fit score percentages against a 400-compound LC/MS/MS spectral library were calculated using an algorithm in which fragment intensity ratios and weighting factors were included. The long-term reproducibility study was conducted using a brand A instrument; after 4 years the reproducibility of the product ion spectra was still 94%, expressed as average Purity score. The inter-laboratory study involved two parts. Firstly, two LC/MS/MS spectral libraries, created independently in separate laboratories using brand A instruments, were compared with each other. The average Fit and Purity scores of spectra from one library against the other were better than 93 and 91%, respectively, when the same collision energies were used. Secondly, for the comparison of product ion spectra between brand A and brand B instruments, fragmentation conditions were first standardized for amitriptyline as the standard analyte. The average Fit scores of brand B spectra against the brand A spectral library varied between 79 and 85% at all three collision energies. These results indicate that, after standardizing the instrumental conditions, LC/MS/MS spectral libraries of drug substances are suitable for inter-laboratory use.