Mass Spectrometric Procedure Research Articles

Chemical Studies of Phytoestrogens and Related Compounds in Dietary Supplements: Flax and Chaparral

High-performance liquid chromatographic (HPLC) and mass spectrometric (MS) procedures were developed to determine lignans in flaxseed (Linum usitatissimum) and chaparral (Larrea tridentata). Flaxseed contains high levels of phytoestrogens. Chaparral has been associated with acute nonviral toxic hepatitis and contains lignans that are structurally similar to known estrogenic compounds. Both flaxseed and chaparral products have been marketed as dietary supplements. A mild enzyme hydrolysis procedure to prevent the formation of artifacts in the isolation step was used in the determination of secoisolariciresinol in flaxseed products. HPLC with ultraviolet spectral (UV) or MS detection was used as the determinative steps. HPLC procedures with UV detection and mass spectrometry were developed to characterize the phenolic components, including lignans and flavonoids, of chaparral and to direct fractionation studies for the bioassays.

Solid-State 13C NMR Analysis of Forage and Byproduct-Derived Fiber and Lignin Residues. Resolution of Some Discrepancies among Chemical, Infrared, and Pyrolysis-Gas Chromatography-Mass Spectroscopic Analyses

Solid-state (13)C NMR was used to investigate changes in the composition of residues from red clover and peanut hulls obtained by four commonly used gravimetric methods for lignin in forages. The samples had also previously been analyzed by infrared and pyrolytic mass spectrometric procedures. The solid-state (13)C NMR spectra enabled the confirmation of a structural interpretation of why the different gravimetric procedures are not chemically equivalent. The three instrumental techniques together enabled a more accurate evaluation of the chemical composition of lignin obtained by each of the four different gravimetric procedures, as well as a comparison of the advantages and limitations of each of the instrumental methods for forage analysis.

A new RaBa chromatographic separation and its application to Ra mass-spectrometric measurement in volcanic rocks

We have adapted the recent chromatographic method described by E.P. Horwitz and coworkers for Sr separation, to perform an efficient BaRa separation for measuring Ra isotopes in volcanic rocks by mass spectrometry. Extraction chromatography is used with a solution of a crown ether ( 4,4′ ( 5′) bis-( tert.-butylcyclohexano)- 18-crown- 6) in 1-octanol, adsorbed on an inert substrate as an extractant. The ElChrom Company markets this resin under the name Sr Spec®. We show that this technique gives a very efficient BaRa separation which compares favourably with previous chromatographic methods. The chemical and mass-spectrometric procedure is described in detail and is applied to Ra measurements in volcanic rocks.

Post-translational modifications in aspartate aminotransferase from Sulfolobus solfataricus. Detection of N-epsilon-methyllysines by mass spectrometry.

Advanced mass spectrometric procedures have been extensively used to provide an accurate structural characterization of aspartate aminotransferase from Sulfolobus solfataricus. The amino acid sequence of this enzyme had previously been deduced from the DNA sequence. The accurate molecular mass of the protein, determined using electrospray mass spectrometry, demonstrated that the amino acid sequence deduced was correct and ruled out the possible presence of large covalent modifications which had been postulated to fit the much higher molecular mass obtained from previous SDS/PAGE experiments. The definition of the entire primary structure of aspartate aminotransferase from S. solfataricus was achieved by exploiting a new mass spectrometric mapping strategy. Initially, the molecular mass of relatively large protein fragments produced by CNBr hydrolysis was accurately determined using electrospray mass spectrometry. The protein regions where structural modifications had occurred were easily identified from their anomalous mass values. The corresponding CNBr fragments were then subdigested with suitable proteases and the resulting peptide mixtures were analysed by fast-atom-bombardment mass spectrometry. This mapping approach led to the detection of two partially modified lysine residues at positions 202 and 384, which had been converted to their N-epsilon-methyl derivatives to a substoichiometric extent.

Measurement of CO2 and HCO3− fluxes in cyanobacteria and microalgae during steady‐state photosynthesis

A mass spectrometric procedure is described which allows the simultaneous estimation of both CO2 and HCO−3 fluxes associated with cyanobacteria and green algae during steady‐stale photosynthesis. This technique utilizes the chemical disequilibrium which exists between CO2 and HCO−3 during photosynthesis in cell suspensions which lack carbonic anhydrase activity. The kinetic equations which are derived for flux determinations are based on models of photosynthesis in both cyanobacteria and green algae which seem most reasonable given our present level of understanding, together with direct measurement of [CO2] estimation of [HCO−3] and application of the kinetic rate constants for the interconversion of CO2 and HCO−3 From measurements made in the light, net uptake of both CO2 and HCO−3 can be readily determined. In addition, analysis of the dark phase immediately following light‐off provides the possibility of also determining the CO2 evolution whích ís occurring during photosynthesis, and thus also the gross CO2 uptake rates in the light. Results are presented for the response of dissolved inorganic carbon (C1) flux rates to external C, in low‐C1 grown cells of both Synechococcus PCC7942 and Chlamydomonas reinhardtii and these are consistent with previous studies showing that such cells possess capacities to utilize both CO2 and HCO−3 for photosynthesis. The advantages and potential errors which are inherent in this technique are discussed together with its potential for future studies on C1 transport under various experimental conditions.

Gas chromatographic—mass spectrometric quantitation of urinary 11-nor-Δ 9-tetrahydrocannabinol-9-carboxylic acid after derivatization by direct extractive alkylation

A gas chromatographic—mass spectrometric procedure for the quantitation of urinary 11-nor-Δ 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) has been developed in which the THC-COOH was derivatized to its corresponding methyl ester—methyl ether derivative by direct extractive alkylation using tetrahexylammonium (THA +) counter-ion and iodomethane dissolved in toluene. The procedure utilised a sample volume of 2 ml and gave a detection limit of 0.25 ng/ml. The inter-run and intra-run coefficients of variation were 7.0% and 4.8%, respectively. The inter-day standard curves were linear in the concentration range 0–300 ng/ml with a mean r = 0.9997 ( n = 4).

Disulfide isoform intermediates in the reoxidation of recombinant human basic fibroblast growth factor

The reoxidation of human recombinant basic fibroblast growth factor was investigated following treatment of the protein with a mixture of reduced and oxidized glutathione, both in the absence and in the presence of protein disulfide isomerase. The oxidative process took place throughout the formation of two transient intermediates and yielded a stable bFGF derivative, GS2-bFGF. All of these components were separated by HPLC and accurately characterized at the molecular level by advanced mass spectrometric procedures. When the reoxidation was carried out in the presence of PDI, a 4-fold increase in the reaction rate was estimated. A mixed disulfide with a single glutathione molecule was shown to occur in the two transient intermediates, each of which has different cysteine residues involved in the linkage. The final product GS2-bFGF was structurally different from other bFGF derivatives previously described [Thompson, S. A. (1992) J. Biol. Chem. 267, 2269-2273; Caccia et al. (1992) Eur. J. Biochem. 204, 649-655]. The four cysteine residues are all involved in disulfide bridges; Cys 34 and Cys 78 are linked to exogenous glutathione, whereas Cys 91 and Cys 101 form an intramolecular S-S bridge.

Bone-lead analysis: development of analytical methodology for milligram samples.

A graphite platform-in-furnace atomic absorption spectrometric (GFAAS) method for determining micrograms/g levels of Pb in 10-20 mg cortical and trabecular primate bone samples is described. The GFAAS method is based on an ambient temperature nitric acid digestion of the sample, matrix modification with palladium nitrate, stabilized temperature platform furnace atomization, and a systematic evaluation of the temperature program of the atomizer. The method of standard additions is mandatory. The detection limit (3 standard deviations of the blank) is 0.19 micrograms Pb/g dry weight for a 10-mg sample taken up in a 1-mL volume. Data are presented on the degree of accuracy and precision. The accuracy of the proposed GFAAS method has been assessed using the IAEA bone reference material, H-5, and by comparing the values obtained in a bone sample with an isotope dilution mass spectrometric (IDMS) procedure. The GFAAS method can be applied to the determination of Pb in 10-20 mg bone samples and for concentrations which are > or = 10 x detection limit. The method is simple, fast, and contamination-free since the entire operation from weighing to GFAAS measurement is carried out in the same centrifuge tube. The method has been applied to the determination of total-Pb in some adult and fetal primate cortical and trabecular bone specimens.

Enantiospecific gas chromatographic—mass spectrometric procedure for the determination of ketoprofen and ibuprofen in synovial fluid and plasma: application to protein binding studies

A method for the enantiospecific quantitation of two commonly prescribed non-steroidal anti-inflammatory drugs (ketoprofen and ibuprofen) is described. The method involves formation of a mixed anhydride of the drug with ethylchloroformate and subsequent conversion to an amide by reaction with optically active amphetamine. The subsequently formed diastereomers are separated by gas chromatography—mass spectrometry using selected-ion monitoring. The assay is capable of quantifying ketoprofen (2 ng/ml) and ibuprofen (3 ng/ml) enantiomers from a 200-μl sample of synovial fluid or plasma and is particularly suitable for protein binding studies.

Molecular weight determination of methyl esters of mycolic acids using thermospray mass spectrometry

Methyl esters of normal fatty acids, corynomycolate and corynomycolenate were used as model compounds for thermospray mass spectrometric procedures for molecular weight determination of the related nocardial mycolic acids. By using ammonium acetate at the positive ion generator, in both cases, a family of ions was produced. The following members were found and corresponded to the adducts: (1) M + H; M + NH 4 and M + H + NH 4 for methyl esters of normal fatty acids, whereas M + H, M + 2H and M + H + NH 4 were the adducts most frequently observed with methyl corynomycolates. The methyl esters of C 40–C 48 mycolic acids from Rhodococcus rhodochrous exhibited prominent peaks corresponding to adducts M + H + NH 4 whereas those corresponding to M + 2H showed slightly lower intensities. The structure M + H had no significant representatives with this subclass of mycolic acids. A similar pattern was observed with methyl esters of C 50–C 54 mycolic acids from Nocardia asteroides GUH-2. Ion peaks C 50–C 54 representing adducts M + 2H and M + H + NH 4 prevailed in the mass spectrum. In this case, the intensities of peaks corresponding to M + 2H were slightly higher than those of the M + H + NH 4. Essentially three main species of nocardomycolic acids were detected: (1) monounsaturated C 50:1, C 52:1 and C 54:1; (2) diunsaturated C 50:2, C 52:2 and C 54:2 and (3) triunsaturated C 52:3 and C 54:3 mycolic acids. The most abundant mycolic acid was C 52:2 followed in decreasing abundance by C 52:1, C 54:2, C 50:2, C 52:3 and C 54:3 mycolic acids.

Mass spectrometric quantification of cysteine-containing leukotrienes in rat bile using 13C-labeled internal standards.

Leukotriene C4 and N-acetyl leukotriene E4 were determined in rat bile using a procedure based on high-performance liquid chromatographic fractionation, hydrogenation to 5-hydroxyeicosanoic acid, and gas chromatographic/mass spectrometric selected ion monitoring analysis of the pentafluorobenzyl ester, trimethylsilyl ether derivatives. 13C4-Labeled analogs of the leukotrienes were synthesized and used as internal standards. The concentrations of both leukotrienes in rat bile were markedly elevated after administration of endotoxin to anesthetized animals; N-acetyl leukotriene E4 was the more abundant product. The presence of leukotriene C4 in rat bile after endotoxin challenge was confirmed by fast atom bombardment/tandem mass spectrometry with precursor ion scanning. Quantitative determination of leukotriene C4 using the tandem mass spectrometric procedure was consistent with the gas chromatographic/mass spectrometric data but the latter procedure gave a substantially lower detection limit.

Characterization of Hemoglobin Lepore Variants by Advanced Mass-Spectrometric Procedures

We describe an analytical protocol for characterizing the molecular structure of hemoglobin (Hb) Lepore variants by using two different mass-spectrometric approaches. The first method consists of direct examination of the chromatographically separated hybrid globins by electro-spray mass spectrometry; the variant Lepore globin is identified through the accurate determination of its molecular mass. Alternatively, the anomalous globins are digested with trypsin and their structures are determined by fast atom bombardment mass-spectrometric analysis of the peptide mixture. The application of this procedure to the identification of Hb Lepore Boston and Hb Lepore Baltimore is described.

Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells.

An important event in the formation of atherosclerotic lesions is the uptake of modified low density lipoprotein (LDL) by macrophages via scavenger receptors. Modification of LDL, which results in its recognition by these receptors, can be initiated by peroxidation of LDL lipids. The first step in this process is the formation of monohydroperoxy derivatives of fatty acids, which are subsequently degraded to the corresponding monohydroxy compounds, or to a variety of secondary oxidation products. In order to understand this process more completely, we have developed a mass spectrometric procedure to measure the amounts of specific hydroperoxy/hydroxy fatty acids formed by oxidation of the major unsaturated fatty acids in human LDL, oleic acid, linoleic acid, and arachidonic acid. Oxidation of human LDL in the presence of a relatively strong stimulus (20 microM CuSO4) resulted in very large increases in the amounts of the major monohydroxy derivatives of linoleic acid (9- and 13-hydroxy derivatives) and arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxy derivatives) in LDL lipids in the early stages of the reaction. After 20 h, the amounts of these products declined due to substrate depletion, but large amounts of monohydroxy derivatives of oleic acid (8-, 10-, and 11-hydroxy derivatives) were detected. Although thiobarbituric acid-reactive substances clearly increased under these conditions, the changes were not nearly so dramatic as those observed for monohydroxy fatty acids. Oxidation of LDL in the presence of endothelial cells, a much milder stimulus, resulted in 2.5- to 3-fold increases in the amounts of monohydroxy derivatives of linoleic and arachidonic acids, as well as thiobarbituric acid-reactive substances, with more modest increases in the amounts of hydroxylated derivatives of oleic acid. There was little positional specificity in the oxidation of the above fatty acids in the presence of either stimulus, suggesting that the formation of these products proceeds primarily by lipid peroxidation, rather than by catalysis by lipoxygenases. However, an important role for lipoxygenases in the initiation of these reactions cannot be excluded. In conclusion, oxidation of LDL in the presence of copper ions or endothelial cells results in the formation of a large number of monohydroxy derivatives of oleic, linoleic, and arachidonic acids. The relative amounts of products formed from each of these fatty acids depends on the strength of the stimulus as well as the incubation time.

FAB overlapping: a strategy for sequencing homologous proteins

Extensive similarity has been shown to exist between the primary structures of closely related proteins from different species, the only differences being restricted to a few amino acid variations. A new mass spectrometric procedure, which has been called FAB-overlapping, has been developed for sequencing highly homologous proteins based on the detection of these small differences as compared with a known protein used as a reference. Several complementary peptide maps are constructed using fast atom bombardment mass spectrometry (FAB-MS) analysis of different proteolytic digests of the unknown protein and the mass values are related to those expected on the basis of the sequence of the reference protein. The mass signals exhibiting unusual mass values identify those regions where variations have taken place; fine location of the mutations can be obtained by coupling simple protein chemistry methodologies with FAB-MS. Using the FAB-overlapping procedure, it was possible to determine the sequence of α 1, α 3 and β globins from water buffalo ( Bubalus bubalis hemoglobins (phenotype AA). Two amino acid substitutions were detected in the buffalo β chain (Lys16 → His and Asn118 → His) whereas the α 1 chains were found the α 1 and α 3 chains were found to contain four amino acid replacements, three of which were identical (Glu23 → Asp, Glu71 → Gly, Phe117 → Cys), and the insertion of an alanine residue in position 124. The only differences between α 1 and α 3 globins were identified in the C -terminal region; α 1 contains a Phe residue at position 130 whereas α 3 shows serine at position 132.

Urinary Markers of Chronic Excessive Ethanol Consumption

A gas chromatographic mass spectrometric procedure is described for the measurement of free ethanol and conjugates of ethanol and acetaldehyde. It was demonstrated that conjugates of ethanol and acetaldehyde were present in urine of alcoholics. Urinary levels of free ethanol and the conjugates in abstaining alcoholics were higher than those of social drinkers. On the average, alcoholics excreted equal amounts of free ethanol and ethanol conjugates while controls eliminated three parts of ethanol conjugates to one part of free ethanol. After 14 days of abstinence, 16/23 (70%) of alcoholics had levels of free ethanol and acetaldehyde conjugates higher than the mean +2 SD of social drinkers for these substances. When the molar ratio of ethanol conjugates/free ethanol was considered, five more subjects who were alcoholics could be classified as being alcoholics. There was one false positive among 46 control subjects.

Biosynthesis of platelet activating factor and 1- O-acyl analogues by endothelial cells

Mass spectrometric procedures have been used to measure 1- O-alkyl-2- O-acetyl-glycero-3-phosphocholine (PAF) and a structural analogue, 1- O-acyl-2- O-acetyl-glycero-3-phosphocholine, biosynthesis in stimulated human umbilical vein endothelial cells (HUVEC). The primary species of acetylated glycerophosphocholine detected were of the 1- O-acyl type, rather than PAF. The amounts of PAF synthesized were much less than reported in earlier studies. In addition, mass spectrometric procedures were used to profile the molecular species of glycerophosphocholine in HUVEC and to determine those species which have arachidonate in the sn-2 position. The pattern of the occurrence of arachidonate in the sn-2 position is very similar to the pattern of synthesis of the acetylated glycerophosphocholines. These results suggest that 1- O-acyl-2- O-acetyl-glycero-3-phosphocholines may have significant but unappreciated biological activities. These results also support the view that glycerophosphocholine molecular species which have arachidonate esterified at the sn-2 position are the immediate precursors for the hydrolysis-acetylation steps which result in the synthesis of sn-2 acetylated glycerophosphocholines, including PAF.

Mass spectrometric analysis of metabolite excretion in five Japanese patients with the late-onset form of glutaric aciduria type II

The variability of clinical and biochemical features in five Japanese patients with the late-onset form of glutaric aciduria type II (GAII) was studied using mass spectrometric procedures. The age at onset ranged from 5 months to five years, presenting acute episodes such as lethargy, hypotonia, hyperammonaemia, hypoglycaemia or Reye's syndrome-like illness, while one of the five cases was asymptomatic at 1 year of age. Organic acid analysis as oxime-trimethylsilyl derivatives by gas chromatography/mass spectrometry revealed the presence of several abnormalities characteristic of GAII in clinically asymptomatic conditions of three patients but not of the two others. Quantitative acylglycine analysis using a stable isotope dilution method and qualitative acylcarnitine analysis by fast atom bombardment mass spectrometry provided diagnostic information in all five patients, regardless of their clinical conditions. However, significant differences in the respective metabolite profiles as well as in their clinical pictures were noted. Although an increased excretion of both isovalerylglycine and isovalerylcarnitine was found in four patients, the fifth showed normal isovalerylglycine excretion during both the acute stage and in remission, despite the increased amount of isovalerylcarnitine in urine. From these results, it was suggested that the variations in clinical severity and metabolite excretion among GAII patients may be attributed not only to the residual enzyme activity at the defective site but also to differences in the capability to conjugate accumulated acyl-coenzyme A.

Quantification of acitretin in human plasma by microbore liquid chromatography—negative chemical ionization mass spectrometry

A highly sensitive liquid chromatographic—mass spectrometric procedure has been developed to quantitate plasma concentrations of acitretin, a dermatologic agent used to treat severe psoriasis. The assay utilizes the combination of normal-phase microbore high-performance liquid chromatography, negative chemical ionization mass spectrometry, selective ion monitoring and stable isotope dilution. The method has been used to measure acitretin and its metabolite, 13- cis-acitretin, over a range of 1–20 ng/ml in human plasma. The inter-assay precision was 5.3% for acitretin and 3.9% for 13- cis-acitretin, while the intra-assay precisions for acitretin and 13- cis-acitretin were 10.8 and 12.7%, respectively. Reproducibility of the assay for acitretin and 13- cis-acitretin, which was determined by the relative standard deviation of multiple analyses of the same quality assurance sample, was 5.9 and 8.1%, respectively.

Determination of plutonium 240/239 ratios in Lake Ontario sediments

A thermal ionization mass spectrometric procedure was developed for determination of 240/239 ratios in subpicogram quantities of plutonium (Pu), and was applied to Pu extracted from Lake Ontario sediment. The detection limit was ∼ 4 fg of Pu and the precision of 240/239 ratio measurement was ∼ 7% in the 0.07–0.42 pg range. Results of sediment analyses at various depths showed in all cases 240/239 ratios near the mean global fallout value of 0.176 ± 0.014. The lack of variability of the ratios with depth negated the use of Pu, present in excess in the near-surface samples due to leakage from a nearby reprocessing plant, as a geochronological meter. The 240/239 ratio of the leakage Pu appeared to be very similar to that of fallout Pu.

Determination of radium isotope ratios and abundances in geological samples by thermal ionization mass spectrometry

We describe chemical separation and mass spectrometric procedures for the measurement of radium isotopes in geologic samples. These methods provide 226Ra/228Ra ratio measurements for 1 g or less of rock sample containing subpicogram amounts of radium with precision better than 1.5% (95% confidence level). Radium-226 concentrations were measured by isotope dilution for smaller sample sizes (100-500 mg) containing as little as 1-10 fg of total 226Ra with similar high precision.