Flow injection with chemical reaction interface-isotope ratio mass spectrometry: an alternative to off-line combustion for detecting low levels of enriched 13C in mass balance studies.

We have evaluated the potential of flow injection chemical reaction interface isotope-ratio mass spectrometry to replace radioactive labeling techniques in material balance studies. A sample is flow injected and transmitted through a desolvation system followed by combustion to form 13CO2 with a microwave-powered chemical reaction interface. We can detect trace amounts of a 13C-labeled drug (3'-azido-3'-deoxythymidine, AZT) in urine or feces. Our ability to quantify less than 100 ng/mL of excess 13C (approximately 1 microgram/mL of 13C-labeled AZT) from a sample equivalent to 10 microL of urine is superior to previous detection limits for 13C in urine that use off-line combustion methods. Parallel studies using 14C-labeled AZT showed that our stable isotope method provides comparable percent excretion data for urine and feces. These results support previous findings that mass balance studies could be carried out with isotope-ratio mass spectrometer, here using doses as low as 1-2 mg/kg.

Element- and isotope-specific detection for high-performance liquid chromatography using chemical reaction interface mass spectrometry.

We have developed a combination of high-performance liquid chromatography (HPLC) and the chemical reaction interface mass spectrometry (CRIMS) method by using a Vestec Universal Interface (UI). This interface provides the extremely high degree of solvent removal that the CRIMS process requires. In doing so, we have produced an HPLC detector with the ability to carry out the element- and isotope-selective analyses with detection that is inherently: linear, structure-independent, sensitive, selective, comprehensive and flexible. The characteristics of the instrumentation and its performance are described.

Direct determination of human urinary cortisol metabolites by HPLC/CRIMS.

Feasibility of using high performance liquid chromatographic input to the chemical reaction interface mass spectrometry system was assessed by measuring the profile of hydrolyzed urinary metabolites of [9,12,12-2H3] cortisol in six human subjects with no preparation other than hydrolysis and solid phase extraction. Relative amounts of tetrahydrocortisol, tetrahydrocortisone, and cortolones (as the sum of alpha- and beta-) were 0.417 +/- 0.047, 0.523 +/- 0.036 and 0.059 +/- 0.019, respectively. The constant reproducibility of the measurements coupled with a profile consistent with that observed by other workers shows that the technique represents an important tool in the determination of metabolites of endogenous molecules.

Importance of mechanistic drug metabolism studies in support of drug discovery: A case study with an N -sulfonylated dipeptide VLA-4 antagonist in rats.

N-(1-(3,5-dichlorobenzenesulfonyl)-2S-methyl-azetidine-2-carbonyl)-L-4-(2',6'-dimethoxyphenyl)phenylalanine (1) is a potent antagonist of the very late activating (VLA) antigen-4. During initial screening, 1 exhibited an apparent plasma clearance (CL) of 227 ml min(-1) kg(-1) in Sprague-Dawley rats following an intravenous bolus dose formulated in an aqueous solution containing 40% polyethylene glycol. Such a high CL value led to speculation that the elimination of compound 1 involved extra-hepatic tissues. However, the apparent plasma CL was reduced to 97 ml in(-1) kg(-1) when a 2-min time point was added to sample collections, and further decreased to 48 ml min(-1) kg(-1) after the dose was formulated in rat plasma. The lung extraction of 1 in rats was negligible whereas the hepatic extraction was > or =90%, based on comparison of area under the curve (AUC) values derived from intra-artery, intravenous, and portal vein administration. In rats dosed intravenously with [(14)C]-1, approximately 91% of the radioactivity was recovered in bile over 48 h, with 85% accounted for in the first 4-h samples. The biliary radioactivity profile consisted of approximately 30% intact parent compound, 20% 1-glucuronide, and 50% oxidation products resulting from O-demethylation or hydroxylation reactions. When incubated with rat liver microsomes, oxidative metabolism of 1 was inhibited completely by 1-aminobenzotriazole (ABT), whereas the oxidation and glucuronidation reactions were little affected in the presence of cyclosporin A (CsA). In contrast, the hepatic extraction of 1 in rats was unperturbed in animals pre-dosed with ABT, but was reduced approximately 60% following treatment with CsA. In vitro, 1 was a substrate of the rat organic anion transporter Oatp1b2, and its cellular uptake was inhibited by CsA. In addition, the hepatic extraction of 1 was approximately 30% lower in Eisai hyperbilirubinaemic rats which lack functional multidrug resistant protein-2 (MRP2). Collectively, these data suggest that the clearance of 1 in rats likely is a result of the combined processes of hepatic oxidation, glucuronidation and biliary excretion, all of which are facilitated by active hepatic uptake of parent compound and subsequent active efflux of both unchanged parent and its metabolites into bile. It was concluded, therefore, that multiple mechanisms contribute to the clearance of 1 in rats, and suggest that appropriate pharmacokinetic properties might be difficult to achieve for this class of compounds. This case study demonstrates that an integrated strategy, incorporating both rapid screening and mechanistic investigations, is of particular value in supporting drug discovery efforts and decision-making processes.

Metabolic activation of a pentafluorophenylethylamine derivative: formation of glutathione conjugates in vitro in the rat.

The aim was to investigate the metabolic activation potential of a pentafluorophenylethylamine derivative (compound I) in vitro in the rat and to identify the cytochrome P450 (CYP) enzymes that catalyse these metabolic activation processes. Reduced glutathione (GSH) was fortified in rat hepatocytes and liver microsomes to trap possible reactive intermediates. Four glutathione conjugates (M1-4) were identified by LC-MS(n) following incubation of compound I in GSH-enriched rat hepatocytes and liver microsomes. Three of these conjugates (M2-4) have not been reported previously for pentafluorophenyl derivatives. Elemental composition analysis of these conjugates was obtained using high-resolution quadrupole time-of-flight mass spectrometry. The formation of GSH conjugate M1 was rationalized as a direct nucleophilic replacement of fluoride by glutathione, whereas the formation of the GSH conjugates M2-4 was proposed to occur by NADPH-dependent metabolic activation of the pentafluorophenyl ring via arene oxide, quinone and/or quinoneimine reactive intermediates. Formation of these conjugates was enhanced three- to five-fold in liver microsomes obtained from phenobarbital- and dexamethasone-treated rats. In incubations with pooled rat liver microsomes and recombinant rat CYP3A1 and CYP3A2, troleandomycin (TAO) reduced the formation of GSH conjugates M2-4 by 80-90%, but it had no effect on the formation of M1. Incubation of compound I with rat supersomes indicated that only CYP3A1 and CYP3A2 were capable of mediating these metabolic activation processes.

Application of high-performance liquid chromatography/chemical reaction interface mass spectrometry for the analysis of conjugated metabolites: a demonstration using deuterated acetaminophen.

The combination of a universal high-performance liquid chromatography/mass spectrometry (HPLC/MS) interface (UI) and the element and isotope-selective capabilities of the chemical reaction interface (CRI) has potential as a comprehensive analysis system for drug conjugates. In this work, we found equal sensitivity for model compounds as their sulfate or glucuronide conjugates. We examined urine and bile samples from Syrian golden hamsters after dosing with (2H4)acetaminophen (D4-APAP), with particular emphasis on the rich range of conjugated metabolites that are known to be produced. Seventeen metabolites were quantified from a single chromatogram of urine; 14 were conjugates. With a combination of authentic standards, selective hydrolysis, and sulfur-selective CRIMS detection, at least partial identification of most of these metabolites was accomplished. The glutathione conjugate of APAP appears the dominant metabolite in bile. The quantitative pattern of APAP metabolism found here is consistent with literature values. It does appear that this HPLC/UI/CRIMS combination has substantial ability to carry out comprehensive metabolite determinations, especially for conjugated species.

Development of an isotope-selective high-performance liquid chromatography detector using chemical-reaction-interface mass spectrometry: application to deuterated cortisol metabolites in urine.

An isotope-selective detector for HPLC based on the particle-beam-interface and the chemical-reaction-interface mass spectrometer (CRIMS) principle is described. This paper focuses on the selective detection of deuterium-labeled analytes. The CRIMS product HD is detected as has been previously described for GC-CRIMS. The analytical performance was not affected by analyte structure or solvent composition. Deuterium detection was linear from 20 ng to 490 ng using 2H3-labeled cortisol. Based on this method, the fractional abundance of three labeled metabolites of cortisol were determined in the urine of a patient infused with tracer amounts of deuterated cortisol.

Determination of benzo[a]pyrene sulfate conjugates from benzo[a]pyrene-treated cells by continuous-flow fast atom bombardment mass spectrometry.

The level of certain water-soluble hydrocarbon conjugates, such as benzo[a]pyrene sulfates (BP-SO4), is a direct measure of carcinogenic polycyclic aromatic hydrocarbon metabolism and an indication of exposure. A new method, based on continuous-flow high-resolution fast atom bombardment mass spectrometry, has been developed for the analysis of BP-SO4 in the medium of cell cultures treated with benzo[a]pyrene. An organic solvent extract of medium from cultures of the human hepatoma cell line (HepG2) was fractionated by reversed-phase SEP-PAK chromatography and microbore high-performance liquid chromatography (HPLC). The HPLC fraction containing BP-SO4 was collected, dried, and injected into a stream of acetonitrile/water/glycerol that was continuously flowing to the tip of the sample probe which was being bombarded continuously by a beam of high-energy xenon atoms. Molecular anions of BP-SO4 (m/z 347) desorbed from the liquid were analyzed by a high-resolution (m/delta m 5000) mass spectrometer and recorded as a function of time. As little as 1.5 pg of BP-SO4 could be detected with a S/N ratio of 8. The mass spectrometer response was linear with respect to the quantity of BP-SO4 injected over the range from 15 to 625 pg. The results obtained with this method show that the HepG2 cultures metabolized 3% of the benzo[a]pyrene into the BP-SO4 conjugate in 24 h. This procedure, which was used to detect and quantify directly BP-SO4 in culture medium without the use of a radiolabeled precursor, should be generally applicable for analyses of sulfated conjugates resulting from the metabolism of different hydrocarbons.

Detection of benzo[a]pyrene sulfate and glucuronide conjugates in cell culture medium by directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry.

An improved method is described for detecting glucuronide and sulfate conjugates of benzo[a]pyrene in medium from cell cultures treated with benzo[a]pyrene. This method is based on a microbore high-performance liquid chromatograph directly coupled to a high-resolution continuous-flow fast atom bombardment mass spectrometer. Sulfate and glucuronide conjugates, as well as some structural isomers of glucuronide conjugates, were fully separated by the reversed-phase microbore high-performance liquid chromatography conditions used in this study. Since the method does not rely on the use of radiolabeled materials, it may be used to detect conjugates of a wide variety of hydrocarbons. The high sensitivity and selectivity of the method were demonstrated by detecting conjugates in the media of cell cultures derived from mice, hamsters and humans.

Studying the reaction between clozapine and glutathione with element-selective detection.

The target of these investigations was a study of covalent binding the antipsychotic drug clozapine and the tripeptide glutathione. Other workers, primarily using radioisotopes, have found many adducts of clozapine and glutathione. We wanted to see how well the chlorine atom in clozapine could serve as an alternate to the use of a radiolabel using the Chemical Reaction Interface/Mass Spectrometer technique with HPLC introduction (HPLC/CRIMS). Here, we examine the capabilities of two such schemes that were previously used with GC introduction: Cl detection with SO2 as the reactant gas; and Cl and S detection using NF3 as the reactant gas. Detecting chlorine as HCl with SO2 was accomplished giving linearity over an 80-fold range of sample size. Incubations of the drug and glutathione with a peroxidase/peroxide system system yielded several metabolites characterized as novel conjugates of clozapine by electrospray mass spectrometry. This tentative identification of two conjugates was confirmed by examining the incubation mixture with NF3 as the CRIMS reactant gas. The simultaneous appearance of both Cl and S is consistent with covalent binding of clozapine to glutathione. A nearly doubled ratio of S to Cl in one peak confirmed the presence of a di-glutathione conjugate. These experiments support our proposition that element selective detection of HPLC effluents with CRIMS can supply additional information, not previously available using radioisotopic methods.

Continuous-flow isotope ratio mass spectrometry using the chemical reaction interface with either gas or liquid chromatographic introduction.

A novel method of sample introduction into an isotope ratio mass spectrometer (IRMS) is described. The technique uses the chemical reaction interface (CRI) to convert samples coming from a gas chromatograph (GC) or high-performance liquid chromatograph (HPLC) into CO2 using a microwave-induced helium plasma. Optimization parameters for both GC/CRI/IRMS and HPLC/CRI/IRMS are described. In both modes of operation, it was possible to obtain 13CO2/12CO2 ratios with standard deviations less than 1%. Investigation of HPLC/CRI/IRMS performance at low and high concentrations (0.5-10 micrograms) resulted in no significant deviations of the isotope ratios. The ability to differentiate samples of different biological origins was illustrated using chlorophyll a from spinach and algae, where a large difference was observed but good precision was maintained (SD < 0.60%).

Isotopic differences in human growth hormone preparations.

The 13C/12C isotope ratios have been measured for human pituitary growth hormone and three commercial growth hormone products in an attempt to differentiate endogenous versus exogenous origin. This might be a strategy to detect doping, as has recently been recognized for testosterone. While all preparations are statistically different from each other, we find that only Humatrope from Lilly has a carbon isotope ratio that is markedly different from those of human growth hormone or Genentech's Nutropin and Protropin. The low renal clearance of growth hormone reduces the applicability of this concept.

Replacing 14C with stable isotopes in drug metabolism studies.

After administration of a mixed dose of both radioisotope and stable-isotope-labeled tirilazad, we carried out a parallel set of HPLC analyses for drug metabolites in bile samples from monkeys and dogs using either radioactivity monitoring (RAM) for 14C or the chemical reaction interface mass spectrometry technique (CRIMS) to detect 13C or 15N. CRIMS is a novel method where analytes are decomposed in a microwave-induced plasma and the elements contained in the analytes are reformulated into small gaseous species that are detected by a mass spectrometer. The comprehensiveness of detection, chromatographic resolution, sensitivity, signal/noise, and quantitative abilities of CRIMS were compared with RAM and in no case was RAM superior. This implies that stable isotopes may be substituted for radioisotopes in studies of drug metabolism where the ability of the latter approach to detect a label independent of the structures in which the label appears has been the primary reason for continuing to use a hazardous and expensive tracer. With HPLC-CRIMS, stable isotopes such as 13C and 15N can be comprehensively detected and quantitative patterns of drug metabolism from biological fluids can be produced that mirror the results when 14C is used.

Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans.

The metabolism of diclofenac has been reported to produce reactive benzoquinone imine intermediates. We describe the identification of mercapturic acid derivatives of diclofenac in rats and humans. Three male Sprague-Dawley rats were administered diclofenac in aqueous solution (pH 7) at 50 mg/kg by intraperitoneal injection, and urine was collected for 24 h. Human urine specimens were obtained, and samples were pooled from 50 individuals. Urine samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Two metabolites with MH(+) ions at m/z 473 were detected in rat urine and identified tentatively as N-acetylcysteine conjugates of monohydroxydiclofenac. Based upon collision-induced fragmentation of the MH(+) ions, accurate mass measurements of product ions, and comparison of LC/MS/MS properties of the metabolites with those of synthetic reference compounds, one metabolite was assigned as 5-hydroxy-4-(N-acetylcystein-S-yl)diclofenac and the other as 4'-hydroxy-3'-(N-acetylcystein-S-yl)diclofenac. The former conjugate also was detected in the pooled human urine sample by multiple reaction-monitoring LC/MS/MS analysis. It is likely that these mercapturic acid derivatives represent degradation products of the corresponding glutathione adducts derived from diclofenac-2,5-quinone imine and 1',4'-quinone imine, respectively. Our data are consistent with previous findings, which suggest that oxidative bioactivation of diclofenac in humans proceeds via benzoquinone imine intermediates.