A solid phase extraction-liquid chromatographic-tandem mass spectrometry method for determination of concentrations of GDC-0941, a small molecule class I phosphatidylinositide 3-kinase inhibitor, to support clinical development.

A solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0941 concentrations in human plasma has been developed and validated to support clinical development. An Oasis MCX 10mg 96-well SPE plate was used to extract plasma samples (50 µL) and the resulting extracts were analyzed using reverse-phase chromatography and mass spectrometer coupled with a turbo-ionspray interface. The method was validated over the calibration curve range 0.500-500 ng/mL with linear regression and 1/x(2) weighting. Within-run relative standard deviation (%RSD) ranged from 1.5 to 11.5%, while the between-run %RSD varied from 0.0 to 4.4%. The accuracy ranged from 96.0% to 110.0% of nominal for within-run and 98.0% to 108.0% of nominal for between-run at all concentrations including the LLOQ quality control at 0.500 ng/mL. Extraction recovery of GDC-0941 was between 79.0% and 86.2%. Stability of GDC-0941 was established in human plasma for 602 days at -70 °C and 598 days at -20°C, respectively, and established in reconstituted sample extracts for 167 h when stored at room temperature. Internal standard normalized matrix factor was 1.1, demonstrating that the use of the stable-labeled internal standard GDC-0941-d(8) effectively compensated observed matrix effect and resulting in no adverse impact on the quality of the data produced. This assay was used for the determination of GDC-0941 human plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.

Determination of GDC-0449, a small-molecule inhibitor of the Hedgehog signaling pathway, in human plasma by solid phase extraction-liquid chromatographic-tandem mass spectrometry.

To support clinical development, a solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0449 concentrations in human plasma has been developed and validated. Samples (200 microl) were extracted using an Oasis MCX 10 mg 96-well SPE plate and the resulting extracts were analyzed using reverse-phase chromatography coupled with a turbo-ionspray interface. The method was validated over calibration curve range 5-5000 ng/mL. Quadratic regression and 1/x(2) weighing were used. Within-run relative standard deviation (%RSD) was within 10.1% and accuracy ranged from 88.6% to 109.0% of nominal. Between-run %RSD was within 8.6% and accuracy ranged from 92.4% to 105.3% of nominal. Extraction recovery of GDC-0449 was between 88.3% and 91.2% as assessed using quality control sample concentrations. GDC-0449 was stable in plasma for 315 days when stored at -70 degrees C and stable in reconstituted sample extracts for 117 h when stored at room temperature. Quantitative matrix effect/ion suppression experiment was performed and no significant matrix ion suppression was observed. This assay allows for the determination of GDC-0449 plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.

Species and gender differences in the formation of an active metabolite of a substituted 2,4-thiazolidinedione insulin sensitizer.

1. The metabolism of a substituted 2,4-thiazolidinedione (P1) with dual PPARalpha/gamma activity was evaluated in male and female rats, dogs and monkeys. A para-hydroxylated metabolite (M1) with potent PPARgamma-selective agonist, was a major circulating drug-related component in female rats, dogs and monkeys, but not in male rats (M1-to-P1 exposure ratio of <1, 3-5, 5 and 5-11 in male rat, monkey, female rat, and dog, respectively). 2. M1 (%) formed in vitro (5, 53, 57-65, 67 and 67% in male rat, monkey, female rat, dog, and human liver microsomes, respectively), rank ordered with M1 (%) formed in vivo (24-45, 53-57, 78, 75-85%, for male rat, monkey, female rat and dog, respectively, after oral administration of P1). 3. The plasma clearance of M1 was higher in male rats (32 ml min(-1) kg(-1) compared with 6, 7 and 2 ml min(-1) kg(-1) in female rat, male monkey and male dogs, respectively). 4. The low amounts of M1 observed in male rats, with the appearance of products of the cleavage of the propyl group between the phenyl groups was probably due to the presence of the sex-specific CYP2C11, which cleaves P1 at the propyl bridge. None of the CYPs present in female rats cleaved P1 at this site and M1 was only produced by CYP2C6. In humans, only CYP2C8 and the polymorphic CYP2C19 produced M1.

Parallel extraction columns and parallel analytical columns coupled with liquid chromatography/tandem mass spectrometry for on-line simultaneous quantification of a drug candidate and its six metabolites in dog plasma.

A method with parallel extraction columns and parallel analytical columns (PEC-PAC) for on-line high-flow liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for simultaneous quantification of a drug candidate and its six metabolites in dog plasma. Two on-line extraction columns were used in parallel for sample extraction and two analytical columns were used in parallel for separation and analysis. The plasma samples, after addition of an internal standard solution, were directly injected onto the PEC-PAC system for purification and analysis. This method allowed the use of one of the extraction columns for analyte purification while the other was being equilibrated. Similarly, one of the analytical columns was employed to separate the analytes while the other was undergoing equilibration. Therefore, the time needed for re-conditioning both extraction and analytical columns was not added to the total analysis time, which resulted in a shorter run time and higher throughput. Moreover, the on-line column extraction LC/MS/MS method made it possible to extract and analyze all seven analytes simultaneously with good precision and accuracy despite their chemical class diversity that included primary, secondary and tertiary amines, an alcohol, an aldehyde and a carboxylic acid. The method was validated with the standard curve ranging from 5.00 to 5000 ng/mL. The intra- and inter-day precision was no more than 8% CV and the assay accuracy was between 95 and 107%.

Use of on-line hydrogen/deuterium exchange to facilitate metabolite identification.

Biotransformation studies performed on an investigational compound (I, represented by R1-CH(NH(2))-CO-N(R2)-CH(2)-S-R3) led to the identification of five metabolites (M1-M5). Based on LC/MS (liquid chromatography/mass spectrometry) analysis which included the use of H(2)O and D(2)O in the mobile phases, they were identified as the sulfoxide (M1), sulfone (M2), carbamoyl glucuronide (M3), N-glucuronide (M4), and N-glucoside (M5) metabolites, respectively. The structure of M3, a less commonly seen carbamoyl glucuronide metabolite, was established using on-line H/D (hydrogen/deuterium) exchange experiments conducted by LC/MS. H/D exchange experiments were also used to distinguish the S-oxidation structures of M1 and M2 from hydroxylation. Herein, the application of deuterium oxide as the LC/MS mobile phase for structural elucidation of drug metabolites in biological matrices is demonstrated.

Ultra-fast gradient vs. fast isocratic chromatography in bioanalytical quantification by liquid chromatography/tandem mass spectrometry.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods developed for quantification using rapid ('ballistic') gradients on narrow bore, short HPLC columns have been previously described by this laboratory. This paper compares the fast gradient approach with the more traditional high-organic isocratic LC/MS/MS methods. The comparison is based on an analysis of the effectiveness of the chromatographic separations when using the two approaches (i.e. k', N, and W). The data presented herein are derived from actual biological samples analyzed as part of the drug discovery process.

Metabolites of caspofungin acetate, a potent antifungal agent, in human plasma and urine.

Caspofungin acetate (MK-0991) is a semisynthetic pneumocandin derivative being developed as a parenteral antifungal agent with broad-spectrum activity against systemic infections such as those caused by Candida and Aspergillus species. Following a 1-h i.v. infusion of 70 mg of [(3)H]MK-0991 to healthy subjects, excretion of drug-related material was very slow, such that 41 and 35% of the dosed radioactivity was recovered in urine and feces, respectively, over 27 days. Plasma and urine samples collected around 24 h postdose contained predominantly unchanged MK-0991, together with trace amounts of a peptide hydrolysis product, M0, a linear peptide. However, at later sampling times, M0 proved to be the major circulating component, whereas corresponding urine specimens contained mainly the hydrolytic metabolites M1 and M2, together with M0 and unchanged MK-0991, whose cumulative urinary excretion over the first 16 days postdose represented 13, 71, 1, and 9%, respectively, of the urinary radioactivity. The major metabolite, M2, was highly polar and extremely unstable under acidic conditions when it was converted to a less polar product identified as N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine gamma-lactone. Derivatization of M2 in aqueous media led to its identification as the corresponding gamma-hydroxy acid, N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine. Metabolite M1, which was extremely polar, eluting from HPLC column just after the void volume, was identified by chemical derivatization as des-acetyl-M2. Thus, the major urinary and plasma metabolites of MK-0991 resulted from peptide hydrolysis and/or N-acetylation.

Bioanalytical applications of 'fast chromatography' to high-throughput liquid chromatography/tandem mass spectrometric quantitation.

A fast chromatographic separation approach that enables rapid method development for high-throughput sample quantification is discussed. This approach has been used to analyze samples from various biological matrices. Data are presented from in vivo pharmacokinetic studies (plasma) and in vitro drug metabolism and transport studies (hepatic microsomes, hepatocytes, and Caco-2 cells).

Determination of in vitro permeability of drug candidates through a caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry.

Studying the permeability of compounds across a Caco-2 cell monolayer is an established in vitro model to screen for oral absorption and to evaluate the mechanism of transport. This assay can also be used to evaluate compounds as potential P-glycoprotein substrates and/or inhibitors. The traditional methods of sample analysis (high-performance liquid chromatography (HPLC) with a UV or fluorescence detector) limit the throughput and sensitivity of this assay. Data are presented here describing the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the analysis of samples derived from the Caco-2 cell studies. During the analysis an automatic switching valve was used to divert the flow from the HPLC column to waste for the first minute, preventing the early eluting salts from entering and contaminating the LC/MS interface. This approach allows the rapid and accurate determination of drug transport across the Caco-2 cell monolayer. The high sensitivity and specificity of LC/MS/MS make this technique an ideal candidate for the low concentration and high throughput routine analysis of Caco-2 cell solutions, especially if multiple compounds are administered and analyzed simultaneously. Thus, the use of LC/MS/MS will increase the value of the Caco-2 cell assay as an in vitro screening tool.

Integrating qualitative and quantitative liquid chromatography/tandem mass spectrometric analysis to support drug discovery.

During the early phase of a drug discovery process, in order to facilitate the selection of drug candidates from a discovery research program, a liquid chromatography tandem mass spectrometry (LC/MS/MS) strategy has been developed to assess the preliminary pharmacokinetics and metabolism of new drug entities. For pharmacokinetic studies using multiple reaction monitoring (MRM), the parent drug concentration and its 'suspected' (predictable) metabolites were monitored in the biological samples simultaneously. For metabolic identification, the general methodologies most frequently used to search for metabolites include full scan, precursor-ion scan, product-ion scan and neutral-loss scan. However, when the metabolites do not produce intense signals for tandem mass spectrometry, a more sensitive and selective assay has to be developed, and MRM would be the method of choice. Likewise, an intelligent guess as to which MRM transitions ought to be used for the metabolites will be considered, based on the product ion mass spectrum of the parent drug.

Disposition of ivermectin and cyclosporin A in CF-1 mice deficient in mdr1a P-glycoprotein.

The pharmacokinetics and hepatic metabolism of [3H] ivermectin (IVM) and [3H]cyclosporin A (CSA) were investigated in a subpopulation of the CF-1 mouse stock naturally deficient in mdr1a p-glycoprotein (PGP). A survey of key drug-metabolizing activities in liver fractions from PGP-deficient (-/-) or wild-type (+/+) animals indicated the two subpopulations are not different in hepatic metabolic activity and capacity. Intravenous pharmacokinetics of CSA were identical between the two groups, and results from microsomal incubations indicated similar biotransformation of IVM and CSA in liver. Intestinal excretion of [3H]IVM and [3H]CSA was enhanced in PGP (+/+) animals. Absence of PGP resulted in higher blood concentrations of IVM after oral dosing, suggesting enhanced absorption of IVM in (-/-) mice. Concentrations of [3H]IVM and [3H]CSA were always greater in the brains of (-/-) mice compared with (+/+) mice after either i.v. or oral administration. In contrast, liver concentrations of either compound were not different between (+/+) and (-/-) animals after an i.v. dose. These results show the PGP (-/-) and (+/+) subpopulations of CF-1 mice are useful for studying the role of mdr1a PGP in systemic exposure and tissue disposition of PGP substrates in the absence of metabolism differences.

Evidence for a novel heme adduct generated by the in vitro reaction of 2,4,6-trinitrotoluene with human hemoglobin using electrospray ionization mass spectrometry.

The bioactivation of nitroaromatic compounds to highly reactive intermediates is responsible for the genotoxic and cytotoxic effects by reaction with DNA and proteins. Due to its continued use as a secondary explosive and its prevalence at contaminated sites, the mechanism of covalent binding of 2,4,6-trinitrotoluene (TNT), or its metabolites, to critical cellular proteins has been of interest. Herein, we report the in vitro reaction of TNT with human hemoglobin under anaerobic and reductive (using sodium hydrosulfite) conditions, yielding a novel adduct between a putative nitrosodinitrotoluene (MW = 211 Da) and the prosthetic heme group (iron protoporphyrin-IX or heme b). While the covalent modification of hemoglobin polypeptide chains by TNT has been established, to our knowledge, this is the first example of a heme-TNT related adduct. This finding could be of relevance in investigation of biotransformation of TNT in subjects exposed to TNT via skin exposure or inhalation.

Effect of cyclodextrins on the hydrolysis of an oxazolidine prodrug of (1R,2S)-(-)-ephedrine-cis-2-(4-methoxyphenyl)-3, 4-dimethyl-5-phenyloxazolidine.

Molecular complexes of an oxazolidine prodrug of (-)-ephedrine, cis-2-(4-methoxyphenyl)-3,4-dimethyl-5-phenyloxazolidine, with alpha-, beta-, dimethyl-beta- (DM-beta-) and hydroxypropl-beta- (HP-beta-) cyclodextrins (CDs) were examined using ionspray mass spectrometry. The results suggest, under our experimental conditions, a 1:1 stoichiometric complex between the prodrug and all CDs. Apart from the putative inclusion complexation, the stabilization effect of CDs on the prodrug in an aqueous solution was studied. beta-, DM-beta- and HP-beta-CD exhibited a retardation effect on the rate of hydrolysis of the prodrug. Conversely, alpha-CD did not alter the rate of hydrolysis even at excess concentrations. Presumably, the larger cavity diameter of beta-CDs may permit a greater depth of penetration of the prodrug, resulting in its shielding from hydrolysis. The observations in this work indicate that mass spectrometry could be a rapid and informative analytical method to aid in the preliminary evaluation of the potential utility of CDs in enhancement of drug stability.

Electrospray mass spectrometry of borane salts: The electrospray needle as an electrochemical cell.

Two borane salts ([(Me)4N][B3H8] and Cs[B3H8]) were examined by electrospray mass spectrometry in the positive ion mode. Acetonitrile solutions provided the most informative spectra; the salts exhibited a remarkable degree of clustering under electrospray conditions, and virtually all signals corresponded to cationic cluster ions of the general formula {[cation (m+)] x [anion (n-)] y }((mx - ny)+). In contrast, methanol solutions of these salts produced only B(OCH3) 4 (-) cluster ions under otherwise identical conditions. (11)B NMR analyses corroborate the identities of the methanol solution species that enter the electrospray source and the reaction product generated during the electrospray process.

Protonation of ferrocene in the gas phase.

Hydrogen-deuterium exchange, proton and deuteron transfer, and collision-induced dissociation experiments involving protonated ferrocene, [Fe(cC5H5)2]H(+), and isotopically labeled analogues have been carried out using a Fourier transform ion cyclotron resonance (FTICR) spectrometer and a double-focusing mass spectrometer of reversed geometry. These experiments reveal that the structure in which the added proton is bound to one of the cyclopentadienyl rings, possibly via agostic interaction with the iron atom, plays an important role in the gas-phase behavior of protonated ferrocene. It is demonstrated that extensive hydrogen atom scrambling occurs in the cyclopentadiene ring and that the extra hydrogen can also switch from one ring to the other, probably via the iron atom. An interpretation is presented which implicates slow thermal unimolecular rearrangement on the FKR time scale from a metal-protonated form to a ring-protonated form which is higher in energy. This interpretation successfully rationalizes the current data as well as previous gas-phase measurements and is found to be in good agreement with solution and matrix isolation studies.

Metastable and collision-induced fragmentation studies of all C4H 12Si (+) isomers; a systematic study of structure-reactivity relations.

Metastable ion (MI) and collision-induced dissociation (CID) mass spectra have been recorded and compared for all nine C4H12Si(+.) isomers. The (Me)4Si(+.), t-BuSiH 3 (+.) , s-BuSiH 3 (+) , and (Me)2EtSiH(+.) isomers have unique MI and CID mass spectra. The MI mass spectra, including the kinetic energy release values, of (Me)(i-Pr)SiH 2 (+.) and (Me)(n-Pr)SiH 2 (+.) are identical, which implies isomerization. MI data also suggest that a fraction of the n-BuSiH 3 (+.) ions rearrange into branched (Me)2EtSiH(+.) ions and a fraction of the n-BuSiH 3 (+.) ions rearrange into branched s-BuSiH 3 (+.) ions. A comparison with the isomeric C5H 12 (+.) pentanes reveals a crucial difference: H2 loss occurs for n-BuSiH 3 (+.) , i-BuSiH 3 (+.) , s-BuSiH 3 (+.) , (Me)(n-Pr)SiH 2 (+.) , (Me)(i-Pr)SiH 2 (+.) , and Et2SiH 2 (+.) , but not for any of the C5Hi 12 (+.) isomers. Generation of four- or five-membered silicon containing rings is suggested for H2 loss from the C4H12Si(+.) silanes.