A comparison of the quantitative methods for the analysis of the platinum-containing anticancer drug [cis-[amminedichloro(2-methylpyridine)]platinum(II)] (ZD0473) by HPLC coupled to either a triple quadrupole mass spectrometer or an inductively coupled plasma mass spectrometer

The use of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICPMS) as means for the quantitative determination of ZD0473, a platinum anticancer drug, and its related biologically active "aqua" compounds in biofluid samples is described. The performance of the resulting HPLC-ICPMS method was compared with that of a conventional HPLC-triple quadrupole mass spectrometer-based (HPLC-MS/MS) system for properties such as limit of detection, linearity, and reproducibility using spiked samples. The methods were then applied to the determination of plasma ultrafitrate concentrations of ZD0473 in dog plasma samples obtained following intravenous and oral administration at 0.5 and 6 mg/kg, respectively. These experiments showed that both methods were capable of providing accurate and precise results but that the HPLC-ICPMS method had advantages of extended linear range and superior sensitivity, providing a limit of quantification of 0.1 ng/mL for ZD0473, as compared to 5 ng/mL using the current HPLC-MS/MS method. In addition, by using a single combined HPLC-ICPMS/MS/MS system, it was possible to determine the relative MS/MS response of the aqua compounds for the first time.     

Sequencing of argentinated peptides by means of electrospray tandem mass spectrometry.

A strategy for semiautomatic sequencing of argentinated (silver-containing) oligopeptides has been developed. Sequencing is based on a search algorithm that identifies a triplet peak relationship in a product ion spectrum of the [M + Ag]+ ion of an oligopeptide. The ions that constitute a triplet are [bn + OH + Ag]+, [bn - H + Ag]+, and [a(n) - H + Ag]+, which are separated by 18 and 28 m/z units, respectively. The difference in the m/z values of adjacent triplets identifies the residue that is "cleaved". Observation of the [yn + H + Ag]+ ion containing the cleaved residue confirms the assignment. Sequencing of argentinated tryptic peptides may prove useful for automated proteome analysis via the sequence tag method.     

Atmospheric pressure covalent adduct chemical ionization tandem mass spectrometry for double bond localization in monoene- and diene-containing triacylglycerols

We report a method to elucidate the structure of triacyl-glycerols (TAGs) containing monoene or diene fatty acyl groups by atmospheric pressure covalent adduct chemical ionization (APCACI) tandem mass spectrometry using acetonitrile as an adduct formation reagent. TAGs were synthesized with the structures ABB and BAB, where A is palmitate (C16:0) and B is an isomeric C18 monoene unsaturated at position 9, 11, or 13 or an isomeric diene unsaturated at positions 9 and 11, 10 and 12, or 9 and 12. In addition to the species at m/z 54 observed in previous CI studies of fatty acid methyl esters, we also found that ions at m/z 42, 81, and 95 undergo covalent reaction with TAGs containing double bonds to yield ions at m/z 40, 54, 81, and 95 units greater than that of the parent TAG: [M + 40]+, [M + 54]+, [M + 81]+, and [M + 95]+ ions. When collisionally dissociated, these ions fragment to produce two or three diagnostic ions that locate the double bonds in the TAG. In addition, ions [RCH=C=O + 40]+ and [RCH=C=O + 54]+ formed from collisional dissociation are of strong abundance in MS/MS spectra, and collisional activation of these ions produces two intense confirmatory diagnostic ions in the MS3 spectra. Fragment ions reflecting neutral loss of an sn-1-acyl group from [M + 40]+ and [M + 54]+ are more abundant than those reflecting neutral loss of an sn-2-acyl group, analogous to previous reports for protonated TAGs. The position of each acyl group on the glycerol backbone is thus determined by the relative abundances of these ions. Under the conditions in our instrument, the [M + 40]+ adduct is at the highest signal and also yields all information about the double bond position and TAG stereochemistry. With the exception of geometries about the double bonds, racemic TAG isomers containing two monoenes or dienes and a saturate can be fully characterized by APCACI-MS/MS/MS.     

Effects of mobile-phase additives, solution pH, ionization constant, and analyte concentration on the sensitivities and electrospray ionization mass spectra of nucleoside antiviral agents

The effects of various mobile-phase additives, solution pH, pKa, and analyte concentration on electrospray ionization mass spectra of a series of purine and pyrimidine nucleoside antiviral agents were studied in both positive and negative ion models. The use of 1% acetic acid resulted in good HPLC separation and the greatest sensitivity for [M + H]+ ions. In the negative ion mode, 50 mM ammonium hydroxide gave the greatest sensitivity for [M - H]- ions. The sensitivities as [M + H]+ ions were significantly larger than the sensitivities as [M - H]- ions for purine antiviral agents. Vidarabine monophosphate and pyrimidine antiviral agents, however, showed comparable or greater sensitivities as [M - H]- ions. The sensitivity as [M + H]+ showed no systematic variation with pH; however, the sensitivity as [M - H]- did increase with increasing pH. At constant pH, the ion intensity of the protonated species increased with increasing pKa. At higher analyte concentrations, dimer (M2H+) and trimer (M3H+) ions were observed. [M + Na]+ adducts were the dominant ions with 0.5 mM sodium salts for these compounds. The spectra of the more basic purine antiviral agents showed no [M + NH4]+ adduct ions, but [M + NH4]+ ions were the major peaks in the spectra of the less basic pyrimidine antiviral agents with ammonium salts. The ammonium adduct ion was formed preferentially when the proton affinity of the analyte was close to that of NH3. Abundant [M + OAc]- ions were observed for all of the antiviral agents except vidarabine monophosphate from solutions with added HOAc, NaOAc, and NH4OAc. The utility of mobile phases containing 1% HOAc or 50 mM NH4OH was demonstrated for chromatographic separations.     

Parent and neutral loss monitoring on a quadrupole ion trap mass spectrometer: screening of acylcarnitines in complex mixtures

A novel and practical technique for performing both parent and neutral loss (P&NL) monitoring experiments on a quadrupole ion trap mass spectrometer is presented. This technique is capable of performing scans analogous to the parent and neutral loss scans routinely applied on tandem-in-space instruments and allows for the screening of a sample to detect analytes of a specific compound class on a chromatographic time-scale. Acylcarnitines were chosen as the model compound class to demonstrate the analytical utility of P&NL monitoring because of their amenability to electrospray ionization (ESI), their unique and informative MS/MS fragmentation pattern, and their importance in biological functions. The [M + H]+ ions of all acylcarnitines dissociate to produce neutral losses of 59 and 161 amu and common product ions at m/z 60, 85, and 144. Both the neutral loss monitoring of 59 amu and the parent ion monitoring of m/z 85 are shown to be capable of identifying acylcarnitine [M + H]+ ions in a synthetic mixture and spiked pig plasma. The neutral loss monitoring of 59 amu is successful in detecting acylcarnitines in an unspiked pig plasma sample.     

Identification of microcystin toxins from a strain of Microcystis aeruginosa by liquid chromatography introduction into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer

The cyclic heptapeptide microcystin toxins produced by a strain of Microcystis aeruginosa that has not been investigated previously were separated by liquid chromatography and identified by high-accuracy m/z measurements of their [M + H]+ ions and the fragment ions produced by collision-activated dissociation of the [M + H]+ ions. The cyanobacteria B2666 strain was cultured in a standard growth medium, and the toxins were released from the cells, extracted from the aqueous phase, and concentrated using standard procedures. The microcystins were separated by reversed-phase microbore liquid chromatography and introduced directly into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer with electrospray ionization. The known microcystins (MC) MC-LR, MC-LA, [MeSer7]MC-LR, MC-LL, MC-LF, and MC-L(Aba) were identified along with the two previously unreported structural variants [Asp3]MC-LA and [Asp3]MC-LL. In addition to the [M + H]+ ions, accurate m/z measurements were made of 12-18 product ions for each identified microcystin. The mean difference between measured and calculated exact m/z was less than 2 parts per million, which often allowed assignment of unique compositions to the observed ions. A mechanism is presented that accounts for an important collision-activated dissociation process that gives valuable sequence ions from microcystins that do not contain arginine. The analytical technique used in this work is capable of supporting fairly rapid and very reliable identifications of known microcystins when standards are not available and of most structural variants independent of additional information from other analytical techniques.     

Analysis of intact tissue by intermediate-pressure MALDI on a linear ion trap mass spectrometer

The use of an intermediate-pressure matrix-assisted laser desorption/ionization (IP-MALDI) source working at 0.17 Torr on a linear ion trap (LIT) was investigated for the analysis of tissue specimens, in particular, spinal cord sections. MALDI, with 2,5-dihydroxybenzoic acid (DHB) as the matrix, was employed for the detection of phospholipids. The matrix was applied to the tissue using electrospray to avoid analyte migration. The results indicate that analyzing tissue specimens at nontraditional MALDI vacuum pressures is possible. Coupling MALDI to an LIT permits the use of MSn, which is critical for the ability to identify compounds desorbed directly from tissue specimens. Using MSn, ions detected from m/z 600-1000 were characterized as phosphatidlycholines, PC. Specifically, using tandem MS, PC ions could be classified as either [M + H]+ or [M + Na]+ because the fragmentation patterns of protonated and sodiated phosphatidlycholines follow different pathways.     

Acetonitrile covalent adduct chemical ionization mass spectrometry for double bond localization in non-methylene-interrupted polyene fatty acid methyl esters

Covalent adduct chemical ionization (CACI) using a product of acetonitrile self-reaction, (1-methyleneimino)-1-ethenylium (MIE; CH2=C=N+=CH2), has been investigated as a method for localizing double bonds in a series of 16 non-methylene-interrupted fatty acid methyl esters (NMI-FAME) of polyenes with three and more double bonds. As with polyunsaturated homoallylic (methylene-interrupted) FAME and conjugated dienes, MIE (m/z 54) reacts across double bonds to yield molecular ions 54 mass units above the parent analyte. [M + 54]+ ions of several 20- and 22-carbon FAME that include one double bond in the C2-C3 position separated by two to five methylene units from a three, four, or five C homoallylic system dissociated according to rules for the homoallylic system, with an additional fragment corresponding to cleavage between the lone double bond and the carboxyl group and defining the position of the lone double bond. Triene FAME with both methylene and ethylene interruption yielded characteristic fragments distinguishable from homoallylic trienes. Fragmentation of fully conjugated trienes in the MS-1 spectra yields ratios of [M + 54]+/[M + 54 - 32]+ (loss of methanol) near unity, which distinguishes them from homoallylic FAME having a ratio of 8 or more; collisionally activated dissociation of [M + 54]+ yields a series of ions, including some rearrangement products, indicative of double bond position. Unlike conjugated dienes, fully conjugated triene diagnostic ion signal ratios did not follow any pattern based on double bond geometry. Partially conjugated trienes behave similarly to monoenes and conjugated dienes, yielding [M + 54]+/[M + 54 - 32]+ of 2-3 and, permitting them to be assigned as partially conjugated FAME using the MS-1 spectrum. They yield unique MS/MS spectra with weaker but assignable fragment ions, along with a diagnostic fragment that locates the lone double bond and permits 6,10,12-octatrienoate to be distinguished from 6,8,12-octatrienoate. The presence of a triple bond did not affect fragment formation in a methylene-interrupted yne-ene but did change fragments in a conjugated yne-ene. These data extend the principle of double bond localization by acetonitrile CACI-MS/MS to double bond structure in complex FAME found in nature.     

Scrubbing ions with molecules: kinetic studies of chemical noise reduction in mass spectrometry using ion-molecule reactions with dimethyl disulfide

The kinetics and product distributions of the reactions of dimethyl disulfide (DMDS) have been investigated with a group of chemical background ions commonly observed in atmospheric pressure ionization (API) mass spectrometry (MS) in order to assess the value of this molecule in filtering (or "scrubbing") these ions by changing their mass/charge (m/z) ratio. The measurements were taken with a novel electrospray ionization/selected ion flow tube/QqQ tandem mass spectrometer. The background ions studied include those with m/z 42 (protonated acetonitrile, ACN), 83 (protonated ACN dimer), 99 (protonated phosphoric acid), 117 (water cluster of m/z 99), 131 (methanol cluster of m/z 99), 149 (protonated phthalic anhydride, formed from the phthalates), and 327 (protonated triphenyl phosphate). In addition, reactions of DMDS have been studied with two model analytes--protonated caffeine and doubly protonated bradykinin--in order to assess the selectivity of DMDS reactivity. All the measurements were taken at 295 +/- 2 K in helium buffer gas at a pressure of 0.35 +/- 0.01 Torr. DMDS was observed to react efficiently with m/z 42 (ACNH+), 149 (from phthalates), and 99 (protonated phosphoric acid), with k/kc=0.91, 0.47, and 0.38, respectively. Only proton transfer was observed with ACNH+, followed by the secondary reaction of [DMDSH]+ with DMDS to yield [CH3S-S(CH3)-SCH3]+. Ligation of DMDS was the dominant primary channel observed for the reaction of the m/z 149 background ion; however, some proton transfer also was observed. Both of these primary product ions react further with DMDS to yield [CH3S-S(CH3)-SCH3]+, the structure of which we have determined computationally using DFT calculations. Only the sequential ligation with two DMDS molecules was observed for the reaction of the m/z 99 ion. Reactions of DMDS with m/z 117 [H3PO4 + H + H2O]+ and m/z 131 [H3PO4 + H + MeOH]+ were observed to proceed with k/kc=0.71 and 0.058, respectively. Ligand substitution of DMDS for H2O predominated ( approximately 94%) over DMDS ligation ( approximately 6%) in the reaction with m/z 117, while only DMDS ligation was observed for the reaction of m/z 131 with DMDS. In contrast, the reactions of DMDS with ions of m/z 83 (protonated dimer of ACN) and 327 (protonated triphenyl phosphate) were extremely inefficient, with k/kc=0.0042 and 0.0079, respectively. The higher reactivity of DMDS toward ACNH+ (m/z 42) compared to (ACN)2H+ (m/z 83) is attributed to the lower proton affinity of the unsolvated ACN. The reactivity of DMDS toward the two model analyte ions studied-protonated caffeine and doubly protonated bradykinin-was negligible, with k/kc=0.0073 and 0.010, for the respective reactions. These results suggest that, under appropriate reagent pressure conditions, DMDS can be an appropriate reagent for chemically filtering out many common API-MS background ions, without significantly affecting the observed intensity of analyte peaks.     

Application of on-line electrochemical derivatization coupled with high-performance liquid chromatography electrospray ionization mass spectrometry for detection and quantitation of (p-chlorophenyl)aniline in biological samples

A rapid, sensitive, and specific assay for detection and quantitation of (p-chlorophenyl)aniline (CPA) in biological samples was developed. The assay was established based on rapid electrochemical oxidation of CPA to a dimerized product (1.0 V vs Pd) with the enhanced detection sensitivity of electrospray mass spectrometer (ES/MS). A "head-to-tail" dimer ([M + H]+ at m/z 217) was exhibited as the predominant species after electrochemical conversion of CPA. Optimal detection sensitivity and specificity for the dimer of CPA that was present in the biological matrix (e.g., rat urine) were achieved through on-line electrochemistry (EC) coupled with high-performance liquid chromatography tandem mass spectrometry. No matrix-associated ion suppression was observed. The limit of detection (S/N approximately 6) was 20 ng/mL, and the limit of quantitation was 50 ng/mL. The calibration curve was exhibited to be quadratic over the range of 50-2000 ng/mL with r2 > 0.99 in various biological matrixes. The assay was validated and used to study the biotransformation of p-chlorophenyl isocyanate (CPIC) to CPA in rats administered intraperitoneally with CPIC (50 mg/kg). The present LC/EC/MS/MS assay of CPA brings important technical advantages to assist in the risk assessment of new chemical entities, which have the potential to produce anilines via biotransformation.     

Identification and characterization of conjugated fatty acid methyl esters of mixed double bond geometry by acetonitrile chemical ionization tandem mass spectrometry

Fatty acids with conjugated double bonds have attracted great interest because of their reported potent bioactivities. However, there are currently no rapid methods for their structural characterization. We report here a convenient mass spectrometry-based strategy to establish double bond geometry by analysis of collisional dissociation products of cis/trans and trans/cis conjugated linoleic acids (CLAs), as methyl esters, and to distinguish CLAs from homoallylic (methylene-interrupted) fatty acids in a single-stage mass spectrum. A series of CLA standards with double bond positions 6,8; 7,9; 8,10; 9,11; 10,12; 11,13; 12,14; and 13,15, with all four possible geometries (cis/trans; trans/cis; cis/cis; trans/trans) were analyzed. The m/z 54 (1-methyleneimino)-1-ethenylium ion, generated by self-reaction of acetonitrile under chemical ionization conditions, reacts with unsaturated fatty acids to yield an [M + 54]+ ion, which decomposes in the single-stage mass spectrum by loss of neutral methanol to form [M + 54 - 32]+. The ratio of [M + 54]+/[M + 54 - 32]+ in the single-stage mass spectra of CLA isomers is 1 order of magnitude less than for homoallylic diene FAME. Collisional dissociation of the [M + 54]+ ion yields two diagnostic ions that contain the alpha- and omega-carbon atoms and is characteristic of double bond position in the analyte. The fragment vinylic to the trans double bond is significantly more abundant than that for the cis double bond, revealing double bond geometry. The ratio of alpha to we diagnostic ion abundances is >4.8 for cis/trans isomers, <0.5 for trans/cis isomers, and 0.7-3.2 for cis/cis and trans/trans isomers. This method provides a rapid alternative to conventional conjugated fatty acid analysis and, together with complementary elution time information provided by gas chromatography, enables rapid, positive identification of double bond position and geometry in most CLA FAME.     

Mass spectrometry and tandem mass spectrometry of citrus limonoids

Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring structure. CAD of the adduct ion [M + H + NH3]+ of limonoid glucosides produced the aglycone moiety corresponding to each glucoside. The combination of mass spectrometry and tandem mass spectrometry provides a powerful technique for identification and characterization of citrus limonoids.     

A validated stable isotope dilution liquid chromatography tandem mass spectrometry assay for the trace analysis of cocaine and its major metabolites in plasma.

A validated method has been developed for the simultaneous quantitation of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, and norcocaine) in rat plasma. The method is based upon the use of stable isotope dilution liquid chromatography/atmospheric pressure chemical ionization/tandem mass spectrometry. Previously reported methods do not have the sensitivity and specificity that can be attained with this method. Plasma samples required no cleanup apart from protein precipitation, and no derivatization was required. Selected reaction monitoring was performed on the transitions of m/z 200 to m/z 182 (ecgonine methyl ester), m/z 290 to m/z 168 (benzoylecgonine), m/z 304 to m/z 182 (cocaine), and m/z 290 to m/z 168 (norcocaine). The standard curves were linear over the range from 2 ng/mL (benzoylecgonine, cocaine, and norcocaine) or 5 ng/mL (ecgonine methyl ester) to 1000 ng/mL in rat plasma. The lower limit of quantitation (LLQ) for benzoylecgonine, cocaine, and norcocaine was 2 ng/mL, and for ecgonine methyl ester, the LLQ was 5 ng/mL for plasma. This simple, rapid, reliable, and sensitive method of quantitation had excellent accuracy and precision for the four analytes. The method was sensitive enough to permit a detailed study of the pharmacokinetics of cocaine and its metabolites after administration of a bolus intravenous dose to rats.     

Analysis of C(14)-C(17) Polychloro-n-alkanes in Environmental Matrixes by Accelerated Solvent Extraction-High-Resolution Gas Chromatography/Electron Capture Negative Ion High-Resolution Mass Spectrometry

A method for quantifying medium-chain (C(14)-C(17)) polychloroalkanes (mPCAs) in environmental matrixes by accelerated solvent extraction high-resolution gas chromatography/electron capture negative ion high-resolution mass spectrometry in the selected ion monitoring mode is presented. The formula group abundance profiles of industrial mPCA mixtures, which are used as standards, and of samples are first determined by monitoring [M - Cl](-) ions of specific m/z values corresponding to the molecular formulas present and by correcting the integrated ion signals for the fractional abundance of the specific m/z value monitored and the number of chlorine atoms in the formula group. mPCA concentrations in environmental samples are then determined by comparing the response of a specific m/z peak in the sample to that in the standard. Extraction recoveries of mPCAs from spiked fish and sodium sulfate (in place of sediment) were >79%. Method detection limits were 13 ng/g for fish and 1.4 ng/μL for sediment, while the analytical detection limit was ～200 pg, at a signal-to-noise ratio of 4:1. By this method, mPCAs were detected in biota and sediment from the mouth of the Detroit River (MI) and ranged from 70 to 900 ng/g. The simultaneous quantitation of C(10)-C(13) (sPCAs) and C(14)-C(17) PCAs, based on monitoring two specific m/z peaks, one characteristic of sPCAs and the other of mPCAs, is also demonstrated.     

On-line LC-MS analysis of urinary porphyrins

A reversed-phase high-performance liquid chromatography-mass spectrometry (LC-MS) method is described for the separation and simultaneous analysis of porphyrins related to disorders of heme biosynthesis (uro-, heptacarboxylic, hexacarboxylic, pentacarboxylic, and coproporphyrins). The method involves initial porphyrin esterification and extraction from urine. Detection and quantification is performed from the extracts by separation with a Hypersil BDS column and on-line detection by MS through coupling with an atmospheric pressure chemical ionization interface. The porphyrin esters are detected as protonated molecules [M + H]+. Their mass spectra also exhibit an [M + Na]+ fragment of lower intensity. The analytical performance of this method is compared with those of LC with UV and fluorescence detection. LC-MS used in selective [M + H]+ ion monitoring provides the lowest detection and quantitation limits. In scan mode, this LC-MS method affords, without further isolation or concentration steps, the measurement of mass spectra of unknown compounds present in the urine of patients with altered porphyrin excretion.     

Liquid chromatography/mass spectrometry methods for distinguishing N-oxides from hydroxylated compounds

This study describes the application of liquid chromatography/mass spectrometry (LC/MS) methods for distinguishing between aliphatic and aromatic hydroxylations and between hydroxylations and N-oxidations. Hydroxylations and N-oxidations are common biotransformation reactions of drugs. Electrospray (ESI) and atmospheric pressure chemical ionization (APCI) were used to generate ions from liquid chromatographic effluents. ESI-MS, ESI-MS/MS, APCI-MS, and APCI-MS/MS experiments were performed on several metabolites and derivatives of loratadine (a long-acting and nonsedating tricyclic antihistamine) using an ion trap mass spectrometer (LCQ) and a triple-quadrupole mass spectrometer (TSQ). The observations are as follows: (1) LC/ESI-MS produced predominantly [M + H]+ ions with minor fragmentation. (2) LC/ESI-MS/MS data, however, showed a predominant loss of water from metabolites with aliphatic hydroxylation while the loss of water was not favored when hydroxylation was phenolic. N-Oxides (aromatic and aliphatic) showed only a small amount of water loss in the MS/MS spectra. (3) Under LC/APCI-MS conditions, aliphatic hydroxylation could be readily distinguished from aromatic hydroxylation based on the extent of water loss. In addition, N-oxides produced distinct [M + H - O]+ ions. These [M + H - O]+ ions were not produced in the APCI-MS spectra of hydroxylated metabolites. (4) Similar to the ESI-MS/MS spectra, the APCI-MS/MS spectra from the (M + H)+ ions of N-oxides yielded a small amount of water loss but no [M + H - O]+ ions. These results indicate that LC/APCI-MS can be used to distinguish between hydroxylated metabolites and N-oxides.     

Direct determination of dibutyl and monobutyl phosphate in a tributyl phosphate/nitric aqueous-phase system by electrospray mass spectrometry

Electrospray ionization mass spectrometry was tested for its potential use in the quantification of monobutyl phosphate (H2MBP) and dibutyl phosphate (HDBP), two degradation products of tributyl phosphate (TBP), the extractant used in the nuclear fuel reprocessing known as the PUREX process. Detection and quantification of these phosphate esters by electrospray in positive and negative ionization mode are reported in this study. This fast and reliable method, which does not require any preliminary sample extraction, appears to be very attractive for process control. Negative ionization mode gave abundant [M - H]- ions for both HDBP and H2MBP products. Thus, the concentration of H2MBP between 0.1 and 10 g/L in concentrated aqueous nitrate solutions can be precisely determined. Moreover, the concentration of HDBP up to 1 g/L in a TBP matrix was evaluated in this mode. For HDBP concentrations below 1 g/L, detection in the positive ionization mode appeared to be attractive. TBP and HDBP cluster detection allowed quantitative HDBP determination. Indeed, small amounts of HDBP in commercial TBP (60 mg/L) could be directly quantified using the specific [2TBP, HDBP + H]+ cluster at m/z 743.     

Low-energy ion--surface reactions of pyrazine with two classes of self-assembled monolayers: influence of alkyl chain orientation

Collisions of pyrazine with two classes of self-assembled monolayer (SAM) films are employed to determine whether surface confinement and the resulting alkyl chain orientation, influences low-energy ion-surface reactions. SAM films formed from n-alkanethiols (CH3(CH2)n-S-Au, n = 14-17) and 4-(4-alkoxyphenylbenzenethiols (4-(4-CH3(CH2)mOC6H4)-C6H4-S-Au, m = 14-17) chemisorbed onto Au (111) substrates are known to exhibit a chain-length-dependent odd-even effect that places the terminal C-C bond into different orientations. Ion-surface collisions (20 eV) of pyrazine molecular ion (M = m/z 80) with these surfaces yield reaction product ions corresponding to the addition of hydrogen atoms ([M + H]+ = m/z 81) and methyl groups ([M + CH3]+ = m/z 95) from the surface to the probe ion. Differences in the relative abundance of the reaction product ions are measured as a function of chain length for both classes of SAM film. SAM films with odd chain lengths (n, m = 14 and 16) have a consistently higher abundance of H addition product ions than SAM films with even chain lengths (n, m = 15 and 17). Alternating reactivity is also observed for the addition of CH3, with methyl addition occurring more readily on even-chain-length films. The variations are consistent with the well-characterized orientation differences known to exist for films of this type. Specifically, odd-chain-length films are oriented such that the last C-C bond is more parallel to the plane of the surface than it is for even-chain-length films. The critical element of the parallel orientation is that it leaves, on average, one hydrogen atom on the terminal methyl and both hydrogen atoms on the first underlying methylene in more reactive positions compared to even chain lengths. Conversely, the trend in the relative abundance of CH3 addition indicates that the orientation produced by an even-chain-length film, with the last C-C bond more perpendicular to the surface, allows the probe ion better access to the methyl carbon. Reflection absorption IR spectroscopy (RAIRS) data independently confirm the orientational disposition of the films. The RAIRS data show that the odd-even effect is less dramatic for the n-alkanethiols when compared to 4-(4-alkoxyphenyl)benzenethiols. A smaller difference in ion-surface reactivity is measured for n-alkanethiols, demonstrating that ion-surface reactions can distinguish subtle differences in average orientation. In short, we report that the extent of ion-surface reactions of pyrazine ion with two classes of SAM films is directed by the spatial orientation of the surface-confined species that participate in the reaction.     

Derivatization of ethinylestradiol with dansyl chloride to enhance electrospray ionization: application in trace analysis of ethinylestradiol in rhesus monkey plasma

The extensive metabolism and administration of low doses of ethinylestradiol (EE) in preclinical animal species necessitates a sensitive analytical method to quantify the drug at low picogram-per-milliliter concentrations in biological matrixes. A highly sensitive and accurate method based on the derivatization of EE with dansyl chloride coupled with liquid chromatography/tandem mass spectrometry is described. The dansyl derivatization of EE introduced a basic secondary nitrogen into the molecule that was readily ionized in commonly used acidic HPLC mobile phases. The derivative showed an intense protonated molecular ion at m/z 530 under positive turbo ion spray ionization. The collision-induced dissociation of this ion formed a distinctive product at m/z 171, corresponding to the protonated 5-(dimethylamino)naphthalene moiety. The selected reaction monitoring, based on the m/z 530 --> 171 transition, was highly specific for EE, since no background signal was observed from blank plasma obtained from rhesus monkeys. The limit of detection, at a signal-to-noise ratio of 5, was 0.2 fg/mL EE spiked into blank plasma. This allowed for a lower limit of quantitation of 5 pg/mL using a 50-microL plasma sample and 10-microL injection of dansylated derivative into the CTC-PAL Leap autosampler coupled to a Sciex API 4000 mass spectrometer. Using fast-gradient liquid chromatography, the analyte peak eluted at 1.6 min. The validation results showed high accuracy (% bias < 4) and precision (% CV < 7.5) at broad linear dynamic ranges (0.005-20 ng/mL), using deuterated EE as internal standard. Therefore, the facile dansyl derivatization coupled with tandem mass spectral analysis allowed the development of a highly sensitive and specific method for quantitation of trace levels of EE in the plasma of rhesus monkeys dosed orally and intravenously with EE.     

Mobility labeling for parallel CID of ion mixtures

An ion mobility/mass spectrometry technique has been developed to record collision-induced dissociation patterns for multiple ions in a parallel fashion. In this approach, a mixture of ions is separated in a drift tube on the basis of differences in mobilities through a buffer gas. As the ions exit the drift tube, they are accelerated into a collision cell and the ensuing fragment ions are dispersed by differences in mass-to-charge (m/z) ratios in a time-of-flight mass spectrometer. Fragment ions that are formed in the collision cell have drift times that are coincident with their antecedent parent ions, allowing the origin of all fragments formed from the mixture of ions to be determined. The approach is demonstrated by examining fragmentation patterns of the [M + H]+ parent and a series of a-, b-, and y-type fragments of [D-Ala2,3]methionine enkephalin.