Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing.

Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled (13)C6 glucose, (13)C3 lactate, and (13)C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.

Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry.

We present here a novel and highly sensitive ion-pair hydrophilic interaction chromatography-tandem mass spectrometry (IP-HILIC-MS/MS) method for quantitation of highly polar acid metabolites like adenine nucleotides. A mobile phase based on diethylamine (DEA) and hexafluoro-2-isopropanol (HFIP) and an aminopropyl (NH2) column were applied for a novel chromatographic separation for the determination of AMP, ADP and ATP in biological matrices. This novel IP-HILIC mechanism could be hypothesized by the ion-pairing reagent (DEA) in the mobile phase forming neutral and hydrophilic complexes with the analytes of polar organic acids. The IP-HILIC-MS/MS assay for adenine nucleotides was successfully validated with satisfactory linearity, sensitivity, accuracy, reproducibility and matrix effects. The lower limit of quantitation (LLOQ) at 2.00ng/mL obtained for ATP showed a least 10-fold higher sensitivity than previous LC-MS/MS assays except nano-LC-MS/MS assay. In summary, this novel IP-HILIC-MS/MS assay provides a sensitive method for nucleotides bioanalysis and shows great potential to determine a number of organic acids in biological matrices.

Ultra sensitive measurement of endogenous epinephrine and norepinephrine in human plasma by semi-automated SPE-LC-MS/MS.

Measurement of endogenous epinephrine (E) and norepinephrine (NE) in human plasma is very challenging due to lower endogenous concentrations as compared with animal plasma. An LC-MS/MS in combination with alumina-based SPE and derivatization procedure was validated for the measurement of E and NE in human plasma with acceptable intra-day and inter-day accuracy and precision. Sample was extracted with semi-automated alumina 96-well solid phase extraction (SPE) cartridge. The resulting eluent was dried and derivatized using d4-acetaldehyde. The analytes were separated on a monolithic C(18) column. Extraction efficiencies were >66% for E and NE. The lower limit of quantitation (LLOQ) was 5.00 pg/mL for E and 20.0 pg/mL for NE.

Simultaneous and high-throughput quantitation of urinary tetranor PGDM and tetranor PGEM by online SPE-LC-MS/MS as inflammatory biomarkers.

Quantitation of urinary tetranor PGDM or tetranor PGEM (tPGDM and tPGEM) in the past was performed separately using off-line SPE LC-MS/MS methods. The manual SPE procedure is generally time-consuming and cost-ineffective. In addition, simultaneous quantitation of tPGDM and tPGEM is favorable yet very challenging because of the similar chemical structures and identical MRM transitions. This work describes the development and validation of a high-throughput online SPE-LC-MS/MS method, allowing simultaneous and high-throughput measurement of tPGDM and tPGEM in human urine. The reportable range of the assay was 0.2-40 ng/ml for tPGDM and 0.5-100 ng/ml for tPGEM. Intra- and inter-assay precision and accuracy determined using quality control samples were all within acceptable ranges (% CV and % Bias < 15%). Tetranor PGDM was stable under all tested conditions while tPGEM was stable at 4 °C and after three F/T cycles but not stable at room temperature for 24 h (recovery below 80%). The assay was applied to measure urinary tPGDM and tPGEM among healthy volunteers, smokers and COPD patients. Significantly higher urinary levels of both tPGDM and tPGEM were observed in COPD patients than those of non-smoking healthy volunteers. These results demonstrated that the high-throughput online SPE-LC-MS/MS assay provides sensitive, reproducible and accurate measurement of urinary tPGDM and tPGEM as biomarkers for assessing inflammatory diseases such as COPD.

Quantification of urinary PGEm, 6-keto PGF(1alpha) and 2,3-dinor-6-keto PGF(1alpha) by UFLC-MS/MS before and after exercise.

Eicosanoids play an important role in the evaluation of pro-inflammatory responses and in the safety and toxicity of novel therapeutic agents. This work describes a high-throughput UFLCMS/MS method for the analysis of three urinary prostanoid biomarkers of pro-inflammatory responses, tetranor PGEm, 6-keto PGF(1alpha) and 2,3-dinor-6-keto PFG(1alpha). Nine male volunteers of various age and fitness level participated in this study. Six provided pre- and post-exercise samples and three provided intraday samples. Tetranor PGEm and 6-keto PGF(1alpha) increased significantly in patients after exercise (p<0.017 and p<0.029). In individual patient sets, tetranor PGEm levels increased from 1.5- to 6-fold pre- vs. post-exercise, levels of 6-keto PGF(1alpha) increased more dramatically from 2- to 55-fold pre- vs. post-exercise. The prostanoid 2,3-dinor-6-keto PGF(1alpha) remained unchanged post-exercise. Data was normalized to urinary creatinine concentration, which increased approximately 40% post-exercise.

Comparison of fused-core and conventional particle size columns by LC-MS/MS and UV: application to pharmacokinetic study.

The chromatographic performance of fused-core (superficially porous) HPLC packing materials was compared with conventional fully porous particle materials for LC-MS/MS analysis of two pharmaceuticals in rat plasma. Two commercially available antidepressants, imipramine and desipramine, were assayed using a conventional analytical C(18) column (5 microm, 2.0 mm x 30 mm) and a fused-core C(18) column (2.7 microm, 2.1 mm x 30 mm). Retention time, column efficiency, pressure drop, resolution, and loading capacity were compared under the same operating conditions. The fused-core column demonstrated reduced assay time by 34% and 2-3-fold increased efficiency (N). Loading capacity up to 25 microl of extract injected on column showed no peak distortion. The registered back-pressure from a flow rate of 1.0 ml/min did not exceed 3400 psi making it compatible with standard HPLC equipment (typically rated to 6000 psi). Two mobile phases were examined, and morpholine as an organic base modifier yielded a 2-5-fold increase in S/N near the limit of detection over triethylamine. The 2.7 microm fused-core column was applied to the analysis of imipramine and desipramine in extracted, protein precipitated rat plasma by LC-MS/MS. The calibration curves were linear in the concentration range of 0.5-1000 ng/ml for both imipramine and desipramine. Intra-run precisions (%CV) and accuracies (%bias) were within +/-7.8% and +/-7.3% at three QC levels and within 14.7% and 14.4% at the LOQ level for both analytes. Following a single method qualification run, the method was applied to the quantitation of pharmacokinetic study samples after oral administration of imipramine to male rats.

Atmospheric pressure desorption/ionization on silicon ion trap mass spectrometry applied to the quantitation of midazolam in rat plasma and determination of midazolam 1'-hydroxylation kinetics in human liver microsomes.

The application of atmospheric pressure desorption/ionization on silicon (AP-DIOS) coupled with ion trap mass spectrometry (ITMS) was investigated for the quantification of midazolam in rat plasma, and determination of midazolam 1'-hydroxylation kinetics in pooled human liver microsomes. Results indicate good sensitivity with absolute detection limits for midazolam in rat plasma of approximately 300 femtograms. A linear dynamic range from approximately 10-5000 ng/mL was obtained in rat plasma with analysis times of 1 min per sample. Kinetic constants for midazolam 1'-hydroxylation in human liver microsomes yielded an apparent Km of 10.0 microM and Vmax of 6.4 nmol/min/mg. Studies investigating the inhibition of 1'-hydroxymidazolam formation by the cytochrome P450 3A4 model inhibitor ketoconazole yielded an IC50 of 0.03 microM. Quantitative precision for replicate analysis of rat plasma and human liver microsomal samples was variable with relative standard deviation (RSD) values ranging from a low of approximately 3% to over 50%, with the highest variability observed in data from human liver microsomal incubations. While preliminary studies investigating the application of AP-DIOS-ITMS suggested feasibility of this technique to typical pharmacokinetic applications, further work is required to understand the underlying causes for the high variability observed in these investigations.

Mechanism-based inactivation of cytochrome P450 2D6 by 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]- 4-[4-(trifluoromethyl)-2-pyridinyl]piperazine: kinetic characterization and evidence for apoprotein adduction.

The kinetics for inactivation of cytochrome P450 2D6 by (1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) were characterized, and the mechanism was determined in an effort to understand the observed time-based inactivation. Loss of dextromethorphan O-demethylase activity following coincubation with EMTPP followed pseudo-first-order kinetics and was both NADPH- and EMTPP-dependent. Inactivation was characterized by an apparent Ki of 5.5 microM with a maximal rate constant for inactivation (kinact) of 0.09 min(-1), a t1/2 of 7.7 min, and a partition ratio of approximately 99. P450 2D6 inactivation was unaffected by coincubation with exogenous nucleophiles or reactive oxygen scavengers and was protected by the competing inhibitors N-4-(trifluoromethyl)benzyl quinidinium bromide and quinidine. After a 30 min incubation with 100 microM EMTPP, dextromethorphan O-demethylase activity was decreased approximately 76%, with a disproportionate loss ( approximately 35%) in carbon monoxide binding. Additional mechanistic studies showed no evidence of either metabolite inhibitory complex formation or heme adduction. However, a P450 2D6 apoprotein adduct was characterized that had a mass shift relative to unadducted P450 2D6 apoprotein consistent with the molecular mass of EMTPP (353 Da). In vitro metabolism studies revealed that EMTPP is susceptible to P450 2D6-mediated hydroxylation and dehydrogenation, postulated to both form via initial hydrogen atom abstraction from the alpha-carbon of the imidazole ethyl substituent. Additional studies demonstrated that while a dehydrogenated EMTPP metabolite was apparently stable and observable, we propose that a thermodynamic partitioning may exist, which results in formation of a second dehydrogenated imidazo-methide-like metabolite that may serve as the reactive species causing mechanism-based inactivation of P450 2D6. Last, trapping studies with EMTPP yielded an N-acetyl cysteine conjugate, which upon tandem MS and NMR analysis revealed adduction to the alpha-carbon of the imidazole ethyl substituent. Overall, evidence suggests that nucleophilic attack of an imidazo-methide-like intermediate by a P450 2D6 active site residue leads to apoprotein adduction and consequent inactivation.