Flavanone absorption after naringin, hesperidin, and citrus administration.

Disposition of citrus flavonoids was evaluated after single oral doses of pure compounds (500 mg naringin and 500 mg hesperidin) and after multiple doses of combined grapefruit juice and orange juice and of once-daily grapefruit. Cumulative urinary recovery indicated low bioavailability ( < 25%) of naringin and hesperidin. The aglycones naringenin and hesperitin were detected in urine and plasma by positive chemical ionization-collisionally activated dissociation tandem mass spectrometry (PCI-CAD MS/MS). After juice administration, PCI-CAD MS/MS detected naringenin, hesperitin, and four related flavanones, tentatively identified as monomethoxy and dimethoxy derivatives. These methoxyflavanones appear to be absorbed from juice. Absorbed citrus flavanones may undergo glucuronidation before urinary excretion.

Internal pulsed valve sample introduction on a quadrupole ion trap mass spectrometer.

A pulsed valve positioned just outside the ion trap electrodes (within the vacuum chamber) has been characterized. The observed gas pulse widths and the maximum ion intensities were found to decrease as the distance between the pulsed valve and the ion trap electrodes increased. An explanation is presented within. The pulsed valve was found to impart temporal separation in ion-molecule reactions by permitting the removal of interfering neutrals. Other factors that affect the degree of temporal separation also are presented.

Differences in the low-energy collision-activated dissociation of carboxylate anions from structurally similar prostaglandins: E2, F 2α, D 2, and DHKF 2α.

Some intriguing discoveries were made concerning the collision-activated dissociation behavior of the derivatized carboxylate anions of PGE2 and PGF2α. The carboxylate anion [MPFB](-) formed from electron-capture negative chemical ionization of the pentafluorobenzyl ester-trimethylsilyl derivative of PGF2α showed little fragmentation under typical collision gas pressures and energies (<2.0 mtorr N2 and <20 eV). In contrast, the daughter spectra of the carboxylate anion of the methoxime-pentafluorobenzyl ester-trimethylsilyl derivative of PGE2 produced many intense fragments under the same conditions.

Negative chemical ionization in quadrupole ion trap mass spectrometry: Effects of applied voltages and reaction times.

The effects of applied voltages and reaction times on negative ion chemical ionization in the quadrupole ion trap are investigated. Mass-selected ejection of undesired reagent ions and selective mass storage of only negative ions are required for practical negative ion chemical ionization. This is achieved by application of rf and dc voltages to the ring electrode to control the mass-to-charge ratios one polarity) of ions stored, as well as by application of a supplemental rf voltage applied across the endcap electrodes to selectively eject ions of a particular mass-to-charge ratio. Even with careful control of these parameters, negative chemical ionization is not as sensitive as electron ionization and positive chemical ionization because of the lack of thermal electrons in the ion trap. Mass selection of the hydroxide anion as a reagent ion and exclusion of all positive ions provide [M - H](-) ions with little or no fragmentation for a wide variety of compounds.

Probing trapped ion energies via ion-molecule reaction kinetics: Quadrupole ion trap mass spectrometry.

We present a detailed study of the energies of the ions stored in a quadrupole ion trap mass spectrometer (QITMS). Previous studies have shown that the rate constant, k, for the charge exchange reaction Ar(+) N(+) 2 →, N(+) 2+Ar increases with increasing ion-molecule center-of-mass kinetic energy (K.E.cm). Thus, we have determined k for this chemical "thermometer" reaction at a variety of Ar and N2 pressures and have assigned K.E.cm values as a function of the q2 of the Ar(+) ion both with and without He buffer gas present in the trap. The K.E.cm energies are found to lie within the range 0.11-0.34 eV over the variety of experimental conditions investigated. Quantitative "cooling" effects due to the presence of He buffer gas are reported, as are increases in K.E.cm due to an increase in the q2 of the Ar(+) ion. "Effective" temperatures of the Ar(+) ions in He buffer are determined based on a Maxwell-Boltzmann distribution of ion energies. The resulting temperatures are found to lie within the range ≈ 1700-3300 K. We have also examined the K.E.cm, values arising from the chemical thermometer reaction of O(+) 2 with CH4, as previous assignments of effective ion temperatures based on this reaction have been called into question.

Alternating RF/DC Isolations for Quantitation with Coeluting Internal Standards in Gas Chromatography/Ion Trap Mass Spectrometry.

A new ion trap scan function for gas chromatography/mass spectrometry (GC/MS) quantitation is described that employs alternating mass-selective storage (rf/dc isolation) of ions from an analyte and its coeluting isotopically labeled internal standard. This scan includes two separate ionization/isolation/mass analysis sequences within the same scan function, each optimized for either the analyte or the internal standard. This results in alternating between analyzing the analyte and the internal standard during their coelution. The method is conceptually similar to using two different scan functions to analyze either the analyte or the internal standard in alternating scans; however, it is much faster because it eliminates the slow procedure of continuously downloading alternating scan functions from disk. This allows more data points to be obtained over a GC peak, resulting in more reproducible GC peak profiles as well as better sensitivity and precision. Results of calibration curves spanning four orders of magnitude (0.5 pg to 5 pg injected on column) obtained by using this method give excellent linear correlations (r (2) > 0.9990) and precision (relative standard deviations of triplicate injections < 10%).

Nonlinear resonance effects during ion storage in a quadrupole ion trap.

Contributions of higher-order fields to the quadrupolar storage field produce nonlinear resonances in the quadrupole ion trap. Storing ions with secular frequencies corresponding to these nonlinear resonances allows absorption of power from the higher-order fields. This results in increased axial and radial amplitudes which can cause ion ejection and collision-induced dissociation (CID). Experiments employing long storage times and/or high ion populations, such as chemical ionization, ion-molecule reaction studies, and resonance excitation CID, can be particularly susceptible to nonlinear resonance effects. The effects of higher-order fields on stored ions are presented and the influence of instrumental parameters such as radiofrequency and direct current voltage (qZ and az values), ion population, and storage time are discussed.

Estimation of mutagenic/carcinogenic potential of environmental contaminants by ion-molecule reactions and tandem mass spectrometry.

The ability to produce and detect products of model DNA/carcinogen ion-molecule reactions is demonstrated in the ion source and the collision cell of a triple quadrupole tandem mass spectrometer. Reaction between adenine and benzoyl chloride in the ion source is shown to produce the DNA adduct benzoyl adenine. The daughter ion mass spectrum of the reaction product is compared to that of the synthesized standard. Mass chromatograms of the reaction between mass-selected pyridine ions and various analytes eluting from a GC column into the collision cell are demonstrated and illustrate the ability to detect only the GC eluates that react with pyridine. This technique could provide a rapid and sensitive method for screening complex environmental samples for carcinogens, as well as for estimating the relative mutagenic/carcinogenic potential of environmental contaminants.

Infrared multiple photon dissociation in the quadrupole ion trap via a multipass optical arrangement.

The design of a novel multipass optical arrangement for use with infrared multiple photon dissociation (IRMPD) in the quadrupole ion trap is presented. This design circumvents previous problems of limited IR laser power, small IR absorption cross sections for many molecules, and the limited ion statistics of trapping and detection of ions for IRMPD in the quadrupole ion trap. In contrast to previous designs that utilized the quadrupole ion store, the quadrupole ion trap was operated in the mass selective instability mode with concurrent resonance ejection. The instrumental design consisted of a modified ring electrode with three spherical concave mirrors mounted on the inner surface of the ring. This modified design allowed for eight laser passes across the radial plane of the ring electrode. IRMPD of protonated bis(2-methoxyethyl)ether (diglyme) was used to characterize the performance of the multipass ring electrode. Two consecutive reactions for the IRMPD of protonated diglyme were observed with a lower energy channel predominant at less than 0.6 J (irradiation times from 1 to 30 ms) and a second channel predominant at energies greater than 0.6 J (irradiation times > 30 ms). Other studies presented include a discussion of the dissociation kinetics of protonated diglyme, the use of a pulsed valve for increased trapping efficiency of parent ion populations, and the effects of laser wavelength and of ion residence time in the radial plane of the ring electrode on photodissociation efficiency.

Pharmaceutical and clinical analysis by tandem mass spectrometry.

Tandem mass spectrometry (MS/MS) is a promising technique for trace determination of compounds in complex mixtures. The application of triple-quadrupole MS/MS to clinical and pharmacological studies has been investigated with major emphasis on rapid screening and determination of drugs and biomolecules in physiological fluids and tissues. These techniques have been applied to a range of problems, including the determination of chlorinated compounds in humans, subpicogram analysis of neurochemicals and the detection of illegal drugs in racing animals. A new technique for determining the structures of all the metabolites of a particular drug has also been developed. It is possible to identify in a single sample all molecular ions which contain substructures characteristic of the parent drug. The structure of each metabolite can then be determined by obtaining the MS/MS spectrum of the molecular ion.

On-line mass spectrometric investigation of the peroxidase-catalysed oxidation of uric acid.

The enzymatic and electrochemical oxidation pathways of uric acid were determined on-line with thermospray-tandem mass spectrometry. Products and intermediates formed as a result of electrooxidation were monitored as the electrode potential was varied. Electrochemical results served as a model for the enzymatic studies. In fact, electrochemical studies were essential for elucidating the structures of intermediates because of the high conversion efficiencies in electrooxidation. Products and intermediates formed as a result of enzymatic oxidation of uric acid were monitored as the reaction time was varied. When the enzymatic oxidation of uric acid with peroxidase and H2O2 was studied, the same intermediates and products were observed as in the electrochemical oxidation. The tandem mass spectrometric results provide convincing evidence that the primary intermediate produced during both the enzymatic and electrochemical oxidation of uric acid has a quinonoid diimine structure. The primary intermediate can follow three distinct reaction pathways to produce the identified final products. The final enzymatic and electrochemical oxidation products observed in these studies were urea, CO2, alloxan, alloxan monohydrate, allantoin, 5-hydroxyhydantoin-5-carboxamide and parabanic acid.

Rapid screening and confirmation for drugs and metabolites in racing animals by tandem mass spectrometry.

A screening and confirmation procedure for drugs and metabolites in the blood serum and urine of racing animals was developed. Equine blood serum was spiked with low concentrations of several drugs of interest. Canine blood serum and urine were collected following oral doses of diethylcarbamazine, procaine, and phenylbutazone. Serum, urine, and extracts of each were analyzed, using a triple quadrupole mass spectrometer. Simultaneous screening of up to 50 drugs was possible in a single sample, in less than 2 minutes. Detection limits for most compounds were in the ng/ml to microgram/ml range, using 1-microliter samples. This procedure provided fast, sensitive screening for selected drugs and metabolites in blood serum and urine.

Interpretation of electrospray/ion trap mass spectra of bile acids and other surfactants.

Electrospray spectra of various bile acids and other surfactants were obtained using an ion trap instrument. Bile acids and bile acid derivatives such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) self associate to form micelles in a stepwise process. Their spectra show a distinct pattern of aggregation, with ions evenly separated in the m/z space. A new parameter, n/z, can be used to characterize such ions, where n is the number of molecules and z is the charge of the aggregate. The values of z were determined using multi-stage mass spectrometry (MS(n)) and high resolution in the ion trap.

Rapid identification of drug metabolites with tandem mass spectrometry.

A method which involves the use of tandem mass spectrometry (MS/MS) for the identification of drug metabolites has been demonstrated with a triple quadrupole mass spectrometer. The method is based on the fact that metabolites usually retain various substructures of the original drug molecule. MS/MS is capable of rapidly identifying molecules with characteristic substructures without prior separation. It is shown that this method makes it possible to postulate possible drug metabolite structures rapidly and systematically without the use of standards. The MS/MS method, as it was applied to the identification of the metabolites of a new antiepileptic drug, zonisamide, is discussed. In this case it was possible to identify isomeric metabolites due to their differences in vaporization times off the probe and their different daughter spectra. The complementary uses of the neutral loss and parent scans for the determination of the site of metabolism is demonstrated. A new figure of merit, the limit of identification, is introduced. The amount of the epoxide metabolite of carbamazepine necessary for its reliable identification in urine was shown to be 0.4 ng/microliter. The application of various techniques to confirm preliminary findings with this MS/MS method are described.

Optimization of short-column gas chromatography/electron ionization mass spectrometry conditions for the determination of underivatized anabolic steroids.

A gas chromatographic/mass spectrometric method based on the use of short capillary gas chromatograph columns (3-5 m) and electron ionization mass spectrometry has been optimized and evaluated for the determination of underivatized anabolic steroids. The short-column gas chromatographic/mass spectrometric method was shown to result in short analysis times and to require minimal sample preparation, but suffered from some loss in sensitivity and chromatographic resolution compared with conventional gas chromatographic/mass spectrometric techniques for derivatized steroids. Therefore, short-column gas chromatographic conditions were optimized to maximize the sample transfer efficiency (sensitivity) from the gas chromatograph into the ion source of the mass spectrometer, while maintaining chromatographic integrity and minimizing thermal decomposition. Mass spectrometric conditions were optimized to maximize ionization efficiency with respect to the intensity of the molecular ion and degree of fragmentation such that positive identification of each steroid could be made based on the resulting mass spectra. Under optimized conditions, we have shown that underivatized anabolic steroids spiked into urine samples can be determined at low-nanogram levels using short-column chromatography/full-scan electron ionization mass spectrometry.

Analysis of human skin emanations by gas chromatography/mass spectrometry. 1. Thermal desorption of attractants for the yellow fever mosquito (Aedes aegypti) from handled glass beads.

Handled glass has the ability to collect and concentrate nonaqueous human skin emanations while minimizing the collection of aqueous perspiration. Compounds originating from the skin and collected on glass have previously been found to attract the Aedes aegypti species of mosquito. Therefore, glass beads were used as the medium to collect skin emanations from humans for subsequent chemical analysis. This process consisted of a 5-15-min collection of sample on glass beads, followed by loading the beads into a gas chromatograph (GC) injector insert for subsequent desorption of the collected compounds onto the GC column. After cryofocusing by liquid nitrogen at the head of the column, the thermally desorbed compounds were analyzed by GC/MS. Microscale purge and trap introduction was also used to provide complementary information. In this case, the beads are held in a round-bottom flask, purged with nitrogen, and heated as the concentrator collects the headspace above the beads. The chromatograms produced by both of these sample introduction methods demonstrate good resolution of a complex sample. Cryofocusing volatiles from handled glass allowed identification of lactic acid, aliphatic fatty acids, and other polar to nonpolar compounds of moderate volatility while purge and trap allowed detection of nonpolar to moderately polar compounds of high volatility.

Characterization of solution-phase and gas-phase reactions in on-line electrochemistry-thermospray tandem mass spectrometry.

Electrochemistry was used on-line with high-performance liquid chromatography-thermospray tandem mass spectrometry to provide insight into the solution-phase decomposition reactions of electrochemically generated oxidation products. Products formed during electrooxidation were monitored as the electrode potential was varied. The solution reactions which follow the initial electron transfer at the electrode are affected by the vaporizer tip temperature of the thermospray probe and the composition of the thermospray buffer. Either hydrolysis or ammonolysis reactions of the initial electrochemical oxidation products can occur with pH 7 ammonium acetate buffer. Both the electrochemically generated and the synthesized disulfide of 6-thiopurine decompose under thermospray conditions to produce 6-thiopurine and purine-6-sulfinate. Solution-phase studies indicate that nucleophilic and electrophilic substitution reactions with purine-6-sulfinate result in the formation of purine, adenine, and hypoxanthine. Products were identified and characterized by tandem mass spectrometry. This work shows the first example of high-performance liquid chromatography used on-line with electrochemistry to separate stable oxidation products prior to analysis by thermospray tandem mass spectrometry. In addition, solution-phase and gas-phase studies with methylamine show that the site of the nucleophilic and electrophilic reactions is probably inside the thermospray probe. Most importantly, these results also show that the on-line combination of electrochemistry with thermospray tandem mass spectrometry provides valuable information about redox and associated chemical reactions of biological molecules such as the structures of intermediates or products as well as providing insight into reaction pathways.