The use of stable isotopes in drug metabolism studies.

Although there is a long history of stable isotopes use in drug metabolism research, it is appropriate to evaluate them in pregnancy drug studies in which safety takes highest priority. It is well established through a number of human and animal experiments that stable isotopes themselves rarely generate additional toxicities beyond the molecules to which they are attached. For the analysis of stable isotopes involved in metabolism studies, mass spectrometry plays the predominant role. Several mass spectrometry-based techniques now exist that enable the selective quantitative detection of stable isotopes with better sensitivity and better retention of chromatographic resolution than do in-line radioactivity monitors for 14C. Even mass balance studies can be performed by using stable isotopes, a type of experiment that still predominantly uses radioisotopes. Some of the newest developments in the use of stable isotopes involve biopolymers, in which fully isotope-labeled species can be generated from cells grown in isotopically labeled growth media. Having shown safety, sensitivity, specificity, and versatility, stable isotopes should play an important role in drug metabolism studies in pregnancy.

Application of high-performance liquid chromatography with isotope-ratio mass spectrometry for measuring low levels of enrichment of underivatized materials.

Combining HPLC separations with an isotope-ratio mass spectrometric (IRMS) detection produces a device capable of measuring very low alterations in 13C abundance from analyte species that cannot be volatilized. Examples are presented showing proteins, carbohydrates, and nucleotides that are eluted from varying types of HPLC columns (reversed-phase, normal-phase, ion-exchange and size-exclusion). This wide range of chromatographic methods enables the analysis of compounds never before amenable to IRMS techniques and may lead to the development of many new assays.

Detection of sulfur-containing impurities in pharmaceutical samples by high performance liquid chromatography/chemical reaction interface mass spectrometry.

This paper describes the use of chemical reaction interface mass spectrometry (CRIMS) combined with liquid chromatography for the detection of trace level sulfur-containing impurities in pharmaceutical materials. A mixture of sulfur- and non-sulfur-containing compounds were analyzed initially to test the system. Then the determination of trace level impurities in a cimetidine drug substance was carried out. Detection of sulfur-containing impurities at less than 0.1% of the major component was obtained with good linearity. The results obtained are consistent with the expected results for this sample and illustrate the applicability of the technique.

Labeling DNA with stable isotopes: economical and practical considerations.

Labeling DNA with stable isotopes to measure cell proliferation can be a technique as effective as 3H-thymidine labeling without the limitations imposed by using radioisotopes. Here, we investigated the relative efficiency of four nonradioactive precursors to DNA: [1-13C]-glycine, [1,2-13C2]-glycine, [U-13C]-glucose, and [U-13C, 15N]-thymidine. The efficiency of incorporation for each of these labeled precursors in HEP G2 cells in culture has been studied. When considering the actual costs of in vivo experiments in which large doses of labeled material are needed, economical constraints may play an important role in defining a practical method. Therefore, the economics of this process were also considered. Using the enrichment per dollar for whichever nucleoside had the highest incorporation in a given experiment, glycine is about five times more economical as a label than thymidine and eight times more economical than glucose in these cells.

An innovative method for measuring hydrogen and deuterium: chemical reaction interface mass spectrometry with nitrogen reactant gas.

A new method for measuring deuterium isotopic enrichment with CRIMS (chemical reaction interface mass spectrometry) is described. Using nitrogen as the reactant gas in a chemical reaction interface generates molecular hydrogen that provides the H2 and HD from which the deuterium content can be analyzed with a benchtop quadrupole mass spectrometer. Samples of deuterated leucine in unlabeled leucine were used as the primary test species. Detection of deuterium enrichment was accurate, precise, and linear. We used this scheme to evaluate the results of a process to acetylate lysine residues in a peptide-neurotensin. With separation on a C18 column, we found a 61% yield of the desired monoethylated product that had a D/H ratio very close to the theoretical one. Isotope ratio monitoring for deuterated species will be important in metabolism studies where CRIMS generates a comprehensive and quantitative view of products of deuterated precursors. Where concerns about metabolic isotope effects of deuterium are absent, the use of deuterium will enable these studies to be performed with simpler syntheses and at less cost than if using 13C or 15N.

Size exclusion chromatography-chemical reaction interface mass spectrometry: "a perfect match".

Size exclusion chromatography (SEC) has been coupled to chemical reaction interface mass spectrometry (CRIMS) for the analysis of biopolymers. This innovative combination allows the analysis of biopolymers with no limitation on the molecular weight and chemical composition of the species under investigation. With SEC-CRIMS we have examined different classes of biopolymers including polynucleotides, proteins, and polysaccharides. Moreover, CRIMS allows the simultaneous detection of multiple organic elements and their stable isotopes. When SEC is interfaced to CRIMS, further information (elemental detection) is obtained, enhancing the analytical capability of SEC. These features have been applied to the detection of a labeled protein (13C-rat growth hormone) in plasma and to the characterization of heparin and low molecular weight heparin from different sources.

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.

A mass spectrum that today is rarely seen.

The mass spectrum of a mixture of H2, He, and N2 with a peak at every mass between m/z 1 and 8 (except for m/z 6) is presented as an example of the richness of information that can exist in this region that is rarely examined in biomedical mass spectrometry. The particular point of the experiment that generated this spectrum was to evaluate a chemical reaction interface mass spectrometric (CRIMS) procedure to measure H/D ratios in labeled analytes. The persistence of an ion-molecule reaction that generates H3+ interferes with the detection of HD+. Observing a second ion-molecule product of hydrogen at m/z 5 leads to a different approach to correcting the signal at m/z 3 than extrapolating the m/z 3/2 ratio to zero hydrogen.

Pharmacokinetic and metabolism studies using uniformly stable isotope labeled proteins with HPLC/CRIMS detection.

We present a novel method for performing pharmacokinetic and metabolism studies on macromolecules that offers advantages over the existing techniques of radiolabeling, immunoassay or bioassays. Our strategy uses macromolecules with stable isotopes uniformly distributed throughout the structure. The stable isotope enrichment is detected using high performance liquid chromatography combined with chemical reaction interface mass spectrometry (HPLC/CRIMS). HPLC/CRIMS is a technique where analytes are first eluted from an HPLC column and then dissociated in a microwave reaction chamber. The dissociated analytes are oxidized using SO2 and the resulting small molecules are detected by the mass spectrometer. The stable-isotope labeled analyte is distinguished from the matrix carbon by monitoring the enrichment of 13CO2. In order to demonstrate the feasibility of performing pharmacokinetic and metabolism studies using this technique, uniformly 13C, 15N-labeled rat growth hormone was administered intravenously to rats and blood samples were collected. Raw plasma samples were analysed by HPLC/CRIMS. Growth hormone was detectable for 1 h following administration. The absolute amounts detected ranged from a high of 66 pmol to a low of 825 fmol in a 20 microL plasma sample. The data were modeled using PCNONLIN and were consistent with a one compartment model. The calculated half-life was 7.7 +/- 0.7 min, with a clearance of 4.5 +/- 0.3 mL min(-1), values consistent with literature reports for growth hormone in rats. No circulating growth hormone metabolites were detected in the plasma. This paper demonstrates a novel technique for performing pharmacokinetic studies of proteins. The uniform labeling strategy also presents a viable comprehensive method for obtaining metabolism data on macromolecules.

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.

Measuring DNA synthesis rates with [1-13C]glycine.

We have devised and evaluated a stable-isotopic method for measuring DNA synthesis rates. The probe is [1-13C]-glycine that is incorporated into purines via de novo biosynthesis. The human hepatoma cell line HEP G2 was grown in medium containing [1-13C]glycine, the cells were harvested at various times, and the DNA was extracted. Following hydrolysis to the nucleosides, a reversed-phase HPLC separation was used to provide separate peaks for deoxythymidine (dT), deoxyadenosine (dA), and deoxyguanosine (dG). The HPLC effluent was continuously fed into a chemical reaction interface and an isotope ratio mass spectrometer (HPLC/CRI/IRMS). The isotope ratio of the CO2 produced in the CRI was used to monitor for enrichment. The cells were grown continuously for 5 days in labeled medium and also in a 1-day pulse labeling experiment where the washout of label was observed for the subsequent 9 days. As predicted from the role of glycine in de novo purine biosynthesis, the isotope ratio of the pyrimidine dT did not change. However, for the two purines, dA and dG, the characteristic log growth behavior of the cells was observed in their 13C/12C ratios and good agreement in the doubling time was obtained for each type of experiment. Parallel experiments that measured the HEP G2 doubling time in culture using tritiated thymidine incorporation and direct cell counts were carried out compare to our new method with established ones. We believe that the use of [1-13C]-glycine and the HPLC/CRI/IRMS is a highly sensitive and selective approach that forms the basis of a method that can measure DNA synthesis rates using a nonradioactive, nontoxic tracer.

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.

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.

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.

Tracing 15N with chemical reaction interface mass spectrometry: a demonstration using 15N-labeled glutamine and asparagine substrates in cell culture.

This research demonstrates how the chemical reaction interface mass spectrometry (CRIMS) approach works for a study of amino acid metabolism in cell culture. 15N-selective chromatograms from both the culture medium and the cytosol of human hepatoma Hep G2 cells that were incubated in the presence of either 12 mM (alpha-15N)glutamine or (alpha-15N)asparagine have been produced. The time course of the distribution of 15N among different amino acids, as well as the enrichment for each amino acid, were observed over a 144 h period. Labeled glutamine was quickly converted into glutamate. After 144 h of incubation, the total amount of 15N was distributed primarily among alanine (50%), proline (28%) and glutamate (21%). The 15N enrichment of alanine and proline reached 44% and 41% respectively. Asparagine was only slowly metabolized by the cells. In addition to the 82% that was retained in asparagine, the remaining 15N in the media at 144 h was found primarily in alanine (8%), glutamate (6.8%) and proline (2.2%). Their enrichments were 20%, 36% and 19% respectively. The minimum detectable amount was 17 pg of 15N entering the CRI. CRIMS appears to be a powerful, facile approach for 15N-tracer experiments.

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.