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%).

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.

The excretion of 7-methyladenine in the urine of rats exposed to carcinogenic methylating agents.

Earlier studies showed that urine of rats which had been injected with the methylating agent N-[3H-methyl]-N-nitrosourea contained a previously undetected metabolic product, 7-[3H-methyl]adenine. This methylpurine, undoubtedly derived from alkylation of nucleic acids followed by depurination, was not labeled when 14C-methyl-labeled methionine was administered concurrently. To establish whether urinary 7-methyladenine (7-MA) might serve as a marker of exposure to exogenous and carcinogenic methylating agents, the excretion of 7-MA following injection of methylating agents was measured. A GC-MS method, using pentafluorobenzyl derivatives and an internal standard of tri-deutero-7-MA, was developed to assay levels of 7-MA. Increasing the i.p. dose of N-methylnitrosourea (MNU) from 2 to 80 mg/kg/rat resulted in a linear increase in urinary 7-MA, which at the highest dose was 1.6 micrograms during the first day and another 0.4 microgram during day 2. Doses of 5 mg/kg MNU led to elevated urinary levels of 7-MA (144 ng) compared to controls (26 ng). Other methylating agents, such as dimethylnitrosamine, N-methyl-N'-nitro-N-nitrosoguanidine and dimethyl sulfate, also provided urinary 7-MA. To determine the fate of injected 7-MA, the administration of 2 micrograms 7-[3H-methyl]adenine led to an 80% recovery of radioactivity in the urine, almost all of it during the first 24 h. No other labeled metabolites were detected. At least for the rat, urinary 7-MA serves as an indicator of exposure to methylating agents.

Quantitation of urinary 7-methyladenine by gas chromatography-mass spectrometry using isotopically labeled internal standards.

We have developed a procedure for isolating and quantifying 7-methyladenine from rat urine following the administration to the rat of methylating agents, such as dimethylnitrosamine. Urinary 7-methyladenine and its trideutero isomer, added as an internal standard, were precipitated with silver nitrate, the precipitate was extracted with HCl, and the extract was further purified by C18-Sep-Pak chromatography. The recovered 7-methyladenine was then derivatized with pentafluorobenzyl bromide at alkaline pH for analysis by gas chromatography-mass spectrometry, indicating a bis(pentafluorobenzyl) conjugate, m/z 509. The mass spectrum of this derivative shows a major fragmentation ion at m/z 328 (and 331 for the trideutero derivative) resulting from the loss of one pentafluorobenzyl group. Levels of urinary 7-methyladenine above 150 pg could be detected from the ratio of the gas chromatography peak areas for these ions, using selective-ion monitoring. The method was selective for the 7-methyl isomer. The procedures developed for the syntheses of deuterated and tritiated 7-methyladenine, which were required for these studies, are also described.

Improved measurement of stable isotope ratios in gas chromatography/mass spectrometry using the microwave-powered chemical reaction interface for mass spectrometry.

The microwave-powered chemical reaction interface for mass spectrometry (CRIMS) has been successfully used for selective detection of analytes labeled with 13C, 15N and D following capillary gas chromatography separation with good analytical characteristics in biological applications. In this study we evaluated how an advanced data system coupled to a quadrupole mass analyzer could improve precision and sensitivity of stable isotope ratio measurements for 13C and 15N. The enrichments of 13C and 15N are determined by monitoring CO2 (m/z 44 and 45) and NO (m/z 30 and 31). These small molecules are produced from the analyte in the chemical reaction interface in the presence of SO2 as a reactant gas. Using caffeine and its 13C1, 15N2-labeled analog for these quantitative studies, we have found that the Vector 2 system improves overall precision (RSD = 0.6% for both carbon and nitrogen) and sensitivity of stable isotope measurements by at least a factor of two compared to the Vector 1 system, and by more than an order of magnitude compared to our older results. With the optimum system we are now able to measure an atom% enrichment of 0.0044 for 15N and 0.015 for 13C in caffeine in the presence of 300 ng of unlabeled material. This is more than half way between isotopic detection limits of conventional gas chromatography/mass spectrometry and the state-of-the-art, which is a gas chromatograph coupled to a chemical combustor and a dual-collector isotope ratio mass spectrometer.

Mass spectrometric analysis of opioid and tachykinin neuropeptides in non-secreting and ACTH-secreting human pituitary adenomas.

In a study to test the hypothesis that defects in the metabolism of neuropeptides might be a contributing factor to human anterior pituitary tumor formation, the proenkephalin A, proopiomelanocortin (POMC), and tachykinin systems, which produce methionine enkephalin (ME), beta-endorphin (BE), and substance P (SP), respectively, were measured in patients who had a wide variety of pituitary tumors. Mass spectrometry was used to optimize the level of molecular specificity of the ME and BE analytical measurements, and radioimmunoassay was used to measure SP-like immunoreactivity (SP-li). Compared to data obtained from pituitaries from post-mortem controls, the non-secreting tumors contained a significantly lower amount of the POMC neuropeptide, BE. The lower ME level was not significant. However, two adrenocorticotrophic hormone (ACTH)-secreting tumors contained ME, BE, and SP-li amounts that were much higher than both the controls and nonsecreting tumors. These data suggest that a hypometabolism of the POMC precursor may be operating in non-secreting tumors, and that a hypermetabolism of the proenkephalin A, POMC, and tachykinin precursors may be operating in two ACTH-secreting tumors. These data demonstrate that mass spectrometry plays a critical role in the study of human pituitary tumors.

Characterization of an aminopeptidase in cerebrospinal fluid. Structure elucidation of enzyme hydrolysis products of synthetic methionine-enkephalin by reversed-phase high-performance liquid chromatography and mass spectrometry.

An aminopeptidase was found in canine cerebrospinal fluid via the presence of two products: Y, which has an [M + H]+ ion at m/z 182; and GGFM, which has an [M + H]+ ion at m/z 411. The linked scan at a constant ratio of the magnetic field to the electric field of the GGFM [M + H]+ ion at m/z 411 generates product ions at m/z 120, 150, 266, 297, 354, 357, and 411. That aminopeptidase was bestatin-sensitive (BSAP = bestatin-sensitive aminopeptidase), and had a half-time for disappearance of 60 min, maximum velocity of 1.08 nmol ml-1 min-1, and Michaelis constant of 0.26 nM.

Mass spectrometric quantification of endogenous beta-endorphin.

Fast atom bombardment (FAB) mass spectrometry and multiple reaction monitoring (MRM) in the B/E linked-field scan mode were used to quantify endogenous beta-endorphin (BE) in individual human pituitary extracts. The experimental protocol includes the addition of a stable isotope-labeled internal standard ((2H4-Ile22)BE1-31, human) to the tissue homogenate before extraction, purification of the native BE by a combination of Sep-Pak chromatography and gradient high-performance liquid chromatography (HPLC), trypsin digestion to cleave BE into smaller peptides, and separation of the tryptic fragment BE20-24 (NAIIK) by isocratic reversed-phase HPLC. Mass spectrometric quantification is based upon recording either (a) the [M + H]+ ions of NAIIK and its deuterated analog ((2H4)NAIIK), or (b) the transitions ([NAIIK + H](+)----[NAI]+) and [((2H4)NAIIK + H](+)----[(2H4)NAI]+) using the B/E linked-field scan. Linear calibration curves were obtained using these two mass spectrometric techniques from standard solutions containing 1.25-20 micrograms of BE; each standard solution also contained 10 micrograms of (2H4)BE. The amounts (means +/- s.d.) of endogenous BE in five separate human pituitaries were found to be 156 +/- 84 [( M + H]+ method) and 169 +/- 99 pmol mg-1 protein (MRM method).

Analysis of methionine enkephalin in human pituitary by multi-dimensional reversed-phase high-performance liquid chromatography, radioreceptor assay, radioimmunoassay, fast atom bombardment mass spectrometry, and mass spectrometry-mass spectrometry.

Methionine enkephalin (ME = YGGFM) was measured in five individual human post-mortem pituitaries using four different analytical methods, with the objective of comparing the molecular specificities of the methods. Radioreceptor assay (RRA) used a receptor-rich preparation from brain and [3H]etorphine as radioligand to determine ME-like receptoractivity (ME-LR). Radioimmunoassay (RIA) measured ME-like immunoreactivity (ME-LI). Pituitary samples analyzed by RRA and RIA were purified first with a high-performance liquid chromatography (HPLC) gradient on a polymer analytical column. Fast atom bombardment mass spectrometry (FAB-MS) in two different detection modes quantified ME using the protonated molecular ion MH+ of ME at 574 a.m.u. and B/E linked-field selected reaction monitoring (SRM) to monitor the specific unimolecular metastable transition that produced the unique amino acid sequence-determining tetrapeptide fragment ion YGGFA+ from the MH+ precursor ion. Both FAB-MS methods used the deuterated internal standard YGG[2H5-F]M. Samples analyzed with FAB-MS were purified first with multi-dimensional reversed-phase HPLC. The first dimension was an ODS gradient, and the second dimension was a polymer isocratic elution. The following ME amounts were measured (mean +/- standard error of the mean): ME-LR, 7.0 +/- 1.9 micrograms g-1 tissue; ME-LI, 1.8 +/- 0.7 micrograms g-1 tissue; MH+, 2.7 +/- 0.6 micrograms g-1 tissue; SRM, 3.0 +/- 0.8 micrograms g-1 tissue. The FAB SRM method provided the highest level of molecular specificity amount these four analytical methods used to measure picomole amounts of endogenous ME in a human pituitary.

Electrospray mass spectrometry for the analysis of opioid peptides and for the quantification of endogenous methionine enkephalin and ß-endorphin.

Electrospray ionization mass spectrometry was used to characterize several different neuropeptides, whose molecular weights ranged from 555 to 3463 Da, and to quantify endogenous methionine enkephalin (ME) and ß -endorphin (ß E) extracted from a human pituitary gland. Methionine enkephalin and leucine enkephalin both yield only an [M + H] + ion with electrospray mass spectrometry; the other peptides produce a series of multiply charged even-electron molecular ions of the general nature [M + nH](n)+ in proportion to the number of basic amino acid units present, with no evidence of fragmentation. The electrospray mass spectra are characterized by low background noise. The quantiftcation of ME is based on a comliarison of the ion current due to the [M + H] + ion of native and of a deuterated ME ([(2)H5 s-(4)Phe]-ME) internal standard. The calibration curve is linear in the range of ca. 1-35 pmol synthetic ME. The amounts of ME determined in three separate human pituitary extracts were 9.1, 8.2, and 4.7 pmol/mg protein. The corresponding amount of ME in a canine pituitary was 39.8 pmol/mg protein. To quantify ß E, the ion current due to the [M + 5H](5) + ion was monitored and compared to an external calibration curve obtained by analyzing solutions of synthetic ß E in the range 5 µmol-50 pmol. The analysis of a human pituitary yielded 660 fmol ß E/mg protein.

Fast atom bombardment mass spectrometric quantitative analysis of methionine-enkephalin in human pituitary tissues.

Picomole amounts of endogenous methionine-enkephalin (ME = YGGFM) were quantified in 11 individual human pituitaries by fast atom bombardment mass spectrometry methods. Quantification was based either upon the comparison of the molecular ion (MH+) current of endogenous ME versus the current of a deuterated ME internal standard (d5-ME) or, similarly, upon the unimolecular decomposition MH+----YGGF-+ In the first field-free region to produce the unique tetrapeptide fragment ion. The latter method used the multiple reaction monitoring (MRM) mode. Native ME was purified with an octadecylsilyl (ODS) disposable cartridge and with multidimensional reversed-phase high-performance liquid chromatography. The amounts of ME determined were 18.26 +/- 19.98 ng of ME/mg of protein with the MH+ method and 15.28 +/- 16.59 ng of ME/mg of protein with the MRM method. A fraction (ca. 4%) of the total amount of ME from one pituitary was used to acquire these quantitative data, and ca. half of the remaining amount of a separate sample (no d5-ME added) was used to obtain a linked scan at constant B/E (B, magnetic field; E, electric field) of the ME MH+ at 574 u to produce the amino acid sequence determining fragment ions at m/z 297, 354, 411, 397, 278, and 425 u corresponding to Y2", Y3", Y4", A4, B3, and B4, respectively. That product ion spectrum was similar to a scan of 100 ng of synthetic ME. We calculated that the amount of pentapeptide for the MRM experiments corresponded to a total of 30 ng (52 pmol) of ME on the probe tip during quantification. On the other hand, we estimated that 3 times more, or 90 ng (156 pmol), ME was on the probe tip during acquisition of the product ion spectrum.