Quantitative analysis of pethidine using liquid secondary ion and tandem mass spectrometry

A method for the quantatitive determination of pethidine in human urine by liquid secondary ion and tandem mass spectrometry is presented. Quantification was carried out by using ketamine as internal standard. It was found that the collision-induced dissociation (CID) spectrum of the [M + H]+ ion of pethidine exhibited a prominent daughter ion at m/z 220 and ketamine also yielded the same daughter ion at m/z 220. For ((quadrupole)) quantitative analysis, the first quadrupole mass filter was set to transmit m/z 220 and a narrow-range magnet scan yielded a spectrum of parents, including m/z 238 and 248, correspending to ketamine and pethidine, respectively.     

Investigation of the electrochemical oxidation products of zotepine and their fragmentation using on-line electrochemistry/electrospray ionization mass spectrometry

When zotepine, an antipsychotic drug, was electrochemically oxidized using electrospray ionization mass spectrometry (ESI-MS) coupled with a microflow electrolytic cell, [M + 16 + H]+ (m/z 348), [M-H]+ (m/z 330) and [M-14 + H]+ (m/z 318) were observed as electrochemical oxidation product ions (M represents the zotepine molecule). Although a major fragment ion that was derived from the dimethyl aminoethyl moiety was observed only at m/z 72 in the collision-induced dissociation (CID) spectrum of zotepine, new fragments such as m/z 315 and 286 ions could be generated in the CID spectrum by combining electrochemical oxidation and CID. Since these fragments were relatively specific with high ion strength, it was thought that they would be useful for developing a sensitive LC-MS/MS assay. The S-oxide and N-demethylated products were detected by electrolysis assuring that a portion of P450 metabolites of zotepine could be mimicked by the electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) system.     

Collision-activated cleavage of a peptide/antibiotic disulfide linkage: possible evidence for intramolecular disulfide bond rearrangement upon collisional activation

Ceftiofur is an important veterinary beta-lactam antibiotic whose bioactive metabolite, desfuroylceftiofur, has a free thiol group. Desfuroylceftiofur (DFC) was reacted with two peptides, [Arg8]-vasopressin and reduced glutathione, both of which have cysteine residues to form disulfide-linked peptide/antibiotic complexes. The products of the reaction, [vasopressin + (DFC-H) + (DFC-H) + H]+, [(vasopressin+H) + (DFC-H) + H]+ and [(glutathione-H) + (DFC-H) + H]+, were analyzed using collision-activated dissociation (CAD) with a quadrupole ion trap tandem mass spectrometer. MS/MS of [vasopressin + (DFC-H) + (DFC-H) + H]+ resulted in facile dissociative loss of one and two covalently bound DFC moieties. Loss of one DFC resulted from either homolytic or heterolytic dissociation of the peptide/antibiotic disulfide bond with equal or unequal partitioning of the two sulfur atoms between the fragment ion and neutral loss. Hydrogen migration preceded heterolytic dissociation. Loss of two DFC moieties from [vasopressin + (DFC-H) + (DFC-H) + H]+ appears to result from collision-activated intramolecular disulfide bond rearrangement (IDBR) to produce cyclic [vasopressin + H]+ (at m/z 1084) as well as other cyclic fragment ions at m/z 1084 +/- 32 and +64. The cyclic structure of these ions could only be inferred as MS/MS may result in rearrangement to non-cyclic structures prior to dissociative loss. IDBR was also detected from MS(3) experiments of [vasopressin + (DFC-H) + (DFC-H) + H]+ fragment ions. MS/MS of [(glutathione-H) + (DFC-H) + H]+ resulted in cleavage of the peptide backbone with retention of the DFC moiety as well as heterolytic cleavage of the peptide/antibiotic disulfide bond to produce the fragment ion: [(DFC-2H) + H]+. These results demonstrate the facile dissociative loss by CAD of DFC moieties covalently attached to peptides through disulfide bonds. Published in 2004 by John Wiley & Sons, Ltd.     

Ion trap mass spectrometry of trimethylsilylamides following gas chromatography

Fatty acid amides are a class of compounds with newly discovered biological activity. The ion trap mass spectrometric characteristics of silylated fatty acid amides were examined. Silylation of primary fatty acid amides is required prior to gas chromatography owing to thermal instability of the underivatized compound. The trimethylsilylated amides do not yield a molecular ion under normal electron ionization conditions (70 eV). With methane as a chemical ionization gas, the [M+H]+ ion appears. The [M+H]+ ion also appears when the helium buffer gas pressure is increased in the ion trap. There are three fragments other than the [M+H]+ peak that are predominant in the ion trap mass spectra of these compounds. Two of the fragments have been reported previously, namely the m/z 59 and the [M-71]+ fragments. The fragment of m/z 72 was identified and is the result of a rearrangement. Isotopic labeling was used to confirm fragment identity and the composition of the rearrangement products. Fragmentation patterns were affected by the amide chain length and concentration.     

Analysis of N7-(benzo[a]pyrene-6-yl)guanine in urine using two-step solid-phase extraction and isotope dilution with liquid chromatography/tandem mass spectrometry

Analysis of urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a DNA adduct induced by benzo[a]pyrene, may serve as a risk-associated biomarker for exposure to polyaromatic hydrocarbons (PAHs). In this study a highly sensitive and specific analytical method, incorporating on-line sample preparation coupled with isotope-dilution liquid chromatography and tandem mass spectrometry (LC/MS/MS), was developed to quantitate this adduct in human urine. In order to achieve accurate quantitation, 15N5-labeled BP-6-N7Gua was synthesized to serve as the internal standard, and a two-step solid-phase extraction (SPE) procedure using C8 and SCX cartridges was used for sample cleanup. BP-6-7-N7Gua was analyzed using positive ion LC/MS/MS operated in multiple reaction monitoring (MRM) mode. The [M+H]+ ions at m/z 402 and 407 and the common fragment ion of [M+H]+ at m/z 252 were monitored for quantification. The recovery of this analyte after two-step SPE was 90%, and the limit of detection was 2.5 fmol/mL in 10 mL of urine. This highly specific and sensitive method for BP-6-N7Gua in urine may be applied to assess exposure to PAHs in coke-oven workers for future molecular epidemiology studies on health effects of PAHs.     

Characterization of moenomycin antibiotics from medicated chicken feed by ion-trap mass spectrometry with electrospray ionization

The antimicrobial moenomycin, commonly used as a growth promoter in livestock, was isolated from medicated chicken feed. The purified extract was subjected to reversed-phase liquid chromatographic separation followed by structural characterization using ion-trap mass spectrometry (ITMS), which allowed identification of five moenomycins (A, A12, C1, C3, and C4) as the major components. The fragmentation patterns of the protonated and deprotonated moenomycin molecules, as well as of a series of sodium adducts, were investigated using ITMS after electrospray ionization. While the protonated molecules [M+H]+ proved highly unstable and underwent extensive in-source fragmentation, isolation and activation of the [M--H]- ions (m/z 1580 for moenomycin-A) yielded simple mass spectra with a dominant base peak corresponding to the loss of the carboxy-glycol and the C25-hydrocarbon chain (m/z 1152 for moenomycin-A). Further study of this fragment ion in an MS3 experiment gave rise to a peculiar product ion (m/z 902 for moenomycin-A) that was attributed to the expulsion of a carbohydrate moiety representing a central building block of the linear molecule. In positive ion mode the generation of the mono-sodiated adduct ions, [M+Na]+, was promoted by amending the mobile phase with 100 microM sodium acetate, but this also resulted in higher adducts of the type [M+2Na--H]+ and [M+3Na--2H]+ arising from the formation of the sodium salts of the phosphate acid diester and subsequently of the carboxylic acid. Substantial differences among the fragment-rich product ion profiles of the three species were observed, and could in part be traced back to the mode of complexation of the additional sodium cation(s).     

Utility of solution electrochemistry mass spectrometry for investigating the formation and detection of biologically important conjugates of acetaminophen

On-line formation and detection of glutathione and cysteine conjugates of acetaminophen were accomplished by the interfacing of a coulometric electrochemical cell with a thermospray mass spectrometer in a flow-injection experiment using a liquid chromatographic pump. Formation of the conjugates occurred only after acetaminophen was oxidized electrochemically by a two-electron transfer to N-acetyl-p-benzoquinoneimine and reacted in a mixing tee with either glutathione or cysteine. The newly formed conjugate was detected by thermospray mass spectrometry by observing the [M + H]+ ion for the acetaminophen-glutathione conjugate at m/z 457, or the [M + H]+ ion for the acetaminophen cysteine conjugate at m/z 271. Both the glutathione and cysteine conjugates produced a common fragment ion at m/z 184. The on-line reaction of glutathione and electrochemically generated N-acetyl-p-benzoquinoneimine was monitored at varying pH. At pH 8.5 the ion intensity for the acetaminophen-glutathione conjugate was greater than at lower pH, indicating that lower proton concentration enhanced the reaction of glutathione with N-acetyl-p-benzoquinoneimine. This on-line electrochemical-thermospray mass spectrometric method demonstrated that acetaminophen conjugates may be formed and detected in the time frame of 1 s.     

Simultaneous measurement of metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivatives in human liver microsomes using liquid chromatography/ion-trap mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method using an atmospheric pressure chemical ionisation source was used to measure the metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivative (1) in human liver microsomes. After 15 min incubation with human liver microsomes, compound 1 exhibited metabolic turnover of 44%. Data-dependent tandem mass spectrometry (MS/MS) scanning was used to generate product ion spectra from the protonated ions of the compound and its metabolites. An unusual metabolite at m/z 407 corresponding to the [M-24+H]+ ion was identified for compound 1. Interestingly, the formation of the [M-24+H]+ ion was not observed in the analogues wherein the fused thieno double bond was substituted (2) and the thieno group replaced by a fused benzo derivative (3). Compounds 2 and 3 exhibited metabolic turnovers of 24 and 30%, yielding oxidative metabolites corresponding to [M+16] and [M+32]+, respectively. Based on these facts the mechanism for [M-24]+ formation in compound 1 through an initial epoxide formation on the double bond of the fused thieno ring followed by hydrolytic ring opening and deacylation is envisaged.     

Application of positive ion chemical ionisation and tandem mass spectrometry combined with gas chromatography to the trace level analysis of ethyl carbamate in bread

A rapid, sensitive and selective method has been developed and validated for the analysis of the contaminant ethyl carbamate (EC) in bread products at the part-per-billion level. The new procedure uses positive ion chemical ionisation (PICI) and tandem mass spectrometry (MS/MS), combined with gas chromatography (GC), on a 'bench-top' triple-quadrupole mass spectrometer. Ammonia was the PICI reagent gas of choice because of its ability to produce abundant [M+H]+ and [M+NH4]+ ions from EC and deuterium-labelled EC (LEC) used as an internal standard. For identification and quantification, selected reaction monitoring (SRM) was used to follow the precursor-to-product ion transitions of m/z 107 --> 90, m/z 107 --> 62 and m/z 90 --> 62 for EC, as well as m/z 112 --> 63 for the LEC internal standard. The limits of detection and quantification were 0.6 and 1.2 microg kg(-1), respectively, and the recovery of the method was 101 +/- 10% at 10 microg kg(-1) and 98 +/- 5% at 100 microg kg(-1). The precision of the method, established under conditions of intermediate reproducibility, did not exceed a relative standard deviation of 7%. The quantitative performance of the new GC/PICI-SRM procedure compared favourably with that of a reference method based on GC/MS and selected ion monitoring (correlation coefficient, r = 0.997). However, the new method had the advantages of reduced sample preparation time, improved sensitivity and unambiguous identification of EC at all concentrations. Application of the new method to the analysis of 50 UK breads showed that levels of EC ranged from 0.6 to 2.3 microg kg(-1) in retail products and from 3.1 to 12.2 microg kg(-1) for breads prepared using domestic breadmaking machines (dry weight basis). Toasting bread in a domestic toaster led to increases of between two- and three-fold in mean EC concentrations.     

Monitoring the degradation of tetracycline by ozone in aqueous medium via atmospheric pressure ionization mass spectrometry

The degradation of tetracycline (1) by ozone in aqueous solution was investigated. High performance liquid chromatography (HPLC), UV-visible spectroscopy (UV-Vis), and total organic carbon (TOC) analyses revealed that although tetracycline was quickly consumed under this oxidative condition, it did not mineralize at all. Continuous monitoring by electrospray ionization mass spectrometry in the positive ion mode, ESI(+)-MS, revealed that tetracycline (1), detected in its protonated form ([1 + H]+) of m/z 445, reacted to yield almost exclusively two unprecedented oxidation products (2 and 3) via a net insertion of one and two oxygen atoms, respectively. Compound 2, suggested to be formed via an initial 1,3-dipolar cycloaddition of ozone at the C11a-C12 double-bond of 1, and Compound 3, proposed to be produced via a subsequent ozone attack at the C2-C3 double-bond of 2, were detected in their protonated forms in the ESI(+)-MS, i.e., [2 + H]+ of m/z 461 and [3 + H]+ of m/z 477, and were further characterized by ESI(+)-MS(n). LC-APCI(+)-MS (liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry in the positive ion mode) experiments corroborated the results.     

多肽研究(Ⅳ)——α-人心房肽片段的快原子轰击质谱(FAB-MS)研究

采用FAB-MS测定α-人心房肽片段的氨基酸序列。通过分析碎片离子的m/z值,可以明确表达各肽段的氨基酸序列。在分子离子区都是[M+H]~+峰,所有片段羰基的α断裂都有肽键断裂和碳碳键断裂两种方式。     

Liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry determination of N-methylpyrrolidinone in riverine biofilms

A rugged procedure utilizing reversed-phase liquid chromatography with positive-ion electrospray ionization mass spectrometry (LC-MS) along with tandem MS is described for the quantification and confirmation of N-methylpyrrolidinone (NMP) in methanolic extracts of riverine biofilm. The LC-MS method provided a 100-fold improvement in detection limits (2 ng g(-1) with a repeatability of 80-95% based on triplicate analyses) compared to a conventional LC-UV detection procedure and was applicable to quantitative analysis of biofilm samples with little or no clean up. Under low-energy collision induced dissociation (CID) conditions (17 V, laboratory frame of reference, with argon as the collision gas), two product-ions of the [M+H]+ ion were formed at m/z 69 [MH-CH3NH2]+ and m/z 58 [MH-CH3NCH]+ with relative abundances of 30% and 5%, respectively. These CID transitions were used to demonstrate that biofilm uptake of a photocatalytically-generated mixture of NMP was rapid once acclimation was achieved.     

Enhancement of atmospheric pressure chemical ionization for the determination of free and glycine-conjugated bile acids in human serum

A highly sensitive and accurate method based on the precolumn derivatization of bile acids (BA) with a high ionization efficiency labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-benzenesulfonate (BDEBS) coupled with LC/MS has been developed. After derivatization, BA molecules introduced a weak basic nitrogen atom into the molecular core structure that was readily ionized in commonly used acidic HPLC mobile phases. Derivatives were sufficiently stable to be efficiently analyzed by atmospheric pressure chemical ionization (APCI)-MS/MS in positive-ion mode. The MS/MS spectra of BA derivatives showed an intense protonated molecular ion at m/z [M + H]+. The collision-induced dissociation of the molecular ion produced fragment ions at [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+. The characteristic fragment ions were at m/z 320.8, 262.8, and 243.7 corresponding to a cleavage of N-CO, O-CO, and C-OCO, respectively, and bonds of derivatized molecules. The selected reaction monitoring, based on the m/z [M+H]+ --> [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+, 320.8, 262.8, and 243.7 transitions, was highly specific for the BA derivatives. The LODs for APCI in a positive-ion mode, at an S/N of 5, were 44.36-153.6 fmol. The validation results showed high accuracy in the range of 93-107% and the mean interday precision for all standards was <15% at broad linear dynamic ranges (0.0244-25 nmol/mL). Good linear responses were observed with coefficients of > 0.9935 in APCI/MS detection. Therefore, the facile BDEBS derivatization coupled with mass spectrometric analysis allowed the development of a highly sensitive and specific method for the quantitation of trace levels of the free and glycine-conjugated BA from human serum samples.     

Fragmentation chemistry of [M + Cu]+ peptide ions containing an N-terminal arginine

[M + Cu]+ peptide ions formed by matrix-assisted laser desorption/ionization from direct desorption off a copper sample stage have sufficient internal energy to undergo metastable ion dissociation in a time-of-flight mass spectrometer. On the basis of fragmentation chemistry of peptides containing an N-terminal arginine, we propose the primary Cu+ ion binding site is the N-terminal arginine with Cu+ binding to the guanidine group of arginine and the N-terminal amine. The principal decay products of [M + Cu]+ peptide ions containing an N-terminal arginine are [a(n) + Cu - H]+ and [b(n) + Cu - H]+ fragments. We show evidence to suggest that [a(n) + Cu - H]+ fragment ions are formed by elimination of CO from [b(n) + Cu - H]+ ions and by direct backbone cleavage. We conclude that Cu+ ionizes the peptide by attaching to the N-terminal arginine residue; however, fragmentation occurs remote from the Cu+ ion attachment site involving metal ion promoted deprotonation to generate a new site of protonation. That is, the fragmentation reactions of [M + Cu]+ ions can be described in terms of a "mobile proton" model. Furthermore, proline residues that are adjacent to the N-terminal arginine do not inhibit formation of [b(n) + Cu - H]+ ion, whereas proline residues that are distant to the charge carrying arginine inhibit formation of [b(n) + Cu - H]+ ions. An unusual fragment ion, [c(n) + Cu + H]+, is also observed for peptides containing lysine, glutamine, or asparagine in close proximity to the Cu+ carrying N-terminal arginine. Mechanisms for formation of this fragment ion are also proposed.     

Measurement of delta 1-tetrahydrocannabinol in plasma to the low picogram range by gas chromatography-mass spectrometry using metastable ion detection

A method for the assay of delta 1-tetrahydrocannabinol (delta 1-THC) in plasma using combined gas chromatography-mass spectrometry with metastable ion monitoring is described. delta 1-THC was extracted with hexane and the extracts were methylated with diazomethane to shift the peaks produced by endogenous plasma constituents away from the cannabinoid region. The delta 1-THC was then converted into its trimethylsilyl derivative and quantitated using the metastable ion at m/z 371 formed in the M+ leads to [M - CH3]+ transition with [1",1",2",2"-2H4]cannabinol as the internal standard. delta 1-THC could be measured to 5 pg/ml in plasma. This assay is 20-100 times more sensitive than existing assays and has the advantage of not needing the usual extensive purification step.     

Analysis of testosterone and dihydrotestosterone from biological fluids as the oxime derivatives using high-performance liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the simultaneous quantitative analysis of dihydrotestosterone (DHT) and testosterone (T) from biological fluids has been developed. Commercially available deuterated analogues were used as internal standards. Steroids were extracted from serum or testicular fluid with hexane/ethyl acetate, evaporated to dryness, and treated with hydroxylamine to form their oxime derivatives. Upon chromatographic separation, the compounds were quantified using selected reaction monitoring (SRM). For T, the [M+H](+) ion at m/z 304 and the fragment ion at m/z 124 were used as the precursor and product ions. For DHT the ion cluster [M+H+ACN](+) at m/z 347 and the dissociated ion [M+H](+) at m/z 306 were used as the precursor and product ions, respectively. The limits of detectability on-column were in the sub-femtomole range for both compounds and the intra-day coefficient of variation (CV) for analysis from serum was less than 7% for both compounds. Given its high reproducibility, sensitivity, and relative simplicity, this assay should be of use in determining androgen levels in biospecimens, particularly in settings where sample quantity or steroid concentration are low.     

Electrospray collision-induced dissociation of testosterone and testosterone hydroxy analogs

Complications with the gas chromatographic analysis of steroids prompted the use of alternative techniques for their identification. High-performance liquid chromatography/mass spectrometry with atmospheric pressure ionization allowed the collection of data for structural identification of these compounds. The objective of this study was to investigate the up-front collision-induced dissociation (UFCID) electrospray ionization (ESI) mass spectra of testosterone and monohydroxylated testosterones. The positive ion UFCID ESI mass spectrum of testosterone showed three significant ions at m/z 97, 109 and 123. The relative abundance of these ions in the UFCID ESI mass spectra of monohydroxylated testosterones varied with the position of the hydroxy group. Statistical data allowed the prediction of hydroxy group position on testosterone by evaluation of the relative abundance of the m/z 97, 109, 121 and 123 ions. Data from the ESI mass spectral analysis of testosterone in a deuterated solvent and from the analysis of cholestenone and 4-androstene-3 beta, 17 beta-diol indicated that the initial ionization of testosterone occurred at the 3-one position. CID parent ion monitoring analyses of the m/z 97, 109 and 123 ions indicated that each resulted from different fragmentation mechanisms and originated directly from the [M + H]+ parent ion. The elemental composition of these fragment ions is proposed based on evidence gathered from the CID analysis of the pseudo-molecular ions of [1,2-2H2]-, [2,2,4,6,6-2H5]-, [6,7-2H2]-, [7-2H]-, [19,19,19-2H3]- and [3,4-13C2]testosterone. The structure and a possible mechanism of formation of the m/z 109 and 123 ions is presented. The results of this study advance the understanding of the mechanisms of collision-induced fragmentation of ions.     

Influence of the labeling group on ionization and fragmentation of carbohydrates in mass spectrometry

The ionization and fragmentation behaviors of carbohydrate derivatives prepared by reaction with 2-aminobenzamide (AB), 1-phenyl-3-methyl-5-pyrazolone (PMP), and phenylhydrazine (PHN) were compared under identical mass spectrometric conditions. It has been shown that the intensities of signals in MS spectra depend on the kind of saccharides investigated and reducing end labels used. PMP sialyllactose, when ionized by ESI/MALDI, produced a mixture of [M + H]+, [M + Na]+, [M - H + 2Na]+ ions in the positive mode and [M - H]-, [M + Na - 2H]- ions in the negative mode. The AB and PHN derivatives formed abundant [M + H]+ and [M - H]- ions in ESI, and by matrix-assisted laser desorption/ionization (MALDI) produced abundant [M + Na]+ ions. PMP- and reduced AB-sialyllactose produced only Y-type fragment ions under both MS/MS sources. In the electrospray ionization (ESI)-MS/MS spectrum of PHN-sialyllactose, abundant ions corresponded to B, Z cleavages and in its MALDI-MS/MS spectrum, the abundant ions were consistent with Y glycosidic cleavages with the concurrence of B, C, and cross-ring fragment ions. In the MALDI-MS spectra of oligosaccharides acquired immediately after derivatization, it was possible to detect only PHN derivatives. After purification, spectra of all three types of derivatives showed high signal-to-noise ratios with the most abundant ions observed for AB reduced saccharides. [M + Na]+ ions were the dominant products and their fragmentation patterns were influenced by the type of the labeling and the kind of oligosaccharide considered. In the MALDI-PSD and -MS/MS spectra of AB-derivatized glycans, higher m/z fragment ions corresponded to B and Y cleavages and the loss of bisecting GlcNAc appeared as a weak signal or was not detected at all. Fragmentation patterns observed in the spectra of hybrid/complex PHN and PMP glycans were more comparable-higher m/z fragments corresponded to B and C glycosidic cleavages. For PHN glycans, the abundance of ions resulting from the loss of bisecting GlcNAc depended on the number of residues linked to the 6-positioned mannose. Also, PHN and PMP derivatives produced cross-ring cleavages with abundances higher than observed in the spectra of AB derivatized oligosaccharides. For high-mannose glycans, the most informative cleavages were provided by AB and PHN type of labeling. Here, PMP produced dominant Y-cleavages from the chitobiose while other ions produced weak signals.     

Quantification of fexofenadine in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry using mosapride as internal standard

A rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of fexofenadine in human plasma using mosapride as internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 1-500 ng/mL for fexofenadine in human plasma. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 5% for fexofenadine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Structural determination of cyclitol derivatives by fast-atom bombardment tandem mass spectrometry

Three cyclitol derivatives were isolated from the marine sponge Sarcotragus sp. by reversed-phase high-performance liquid chromatography and analyzed by fast-atom bombardment mass spectrometry (FAB-MS). Their structural elucidation was carried out with FAB tandem mass spectrometry (FAB-MS/MS). FAB-MS spectra produced a significant abundance of the sodium adducts [M+Na]+ and [M+2Na-H]+ from a mixture of m-NBA and NaI. In addition, trifluoroacetylation of the cyclitol derivatives was used for confirmation of the presence of the cyclitol ring. High abundance [M-5H+5CF3CO+Na]+ ions were observed in the FAB-MS spectra of the trifluoroacetyl-cyclitol derivatives. Collision-induced dissociation (CID) of the [M+Na]+ ions produced diverse product ions via a series of dissociative processes. Charge-remote fragmentation (CRF) patterns of [M+Na]+ ions were very useful for the identification of product ions which are characteristic for the cyclitol ring and long hydrocarbon chains substituted at the glycerol backbone. Moreover, the CID-MS/MS spectra of the [M+Na]+ ions yielded characteristic product ions at m/z 53, 83, 113, 155 and 171 for the cyclitol moiety, and at m/z 213, 229 and 245 for the glycerol backbone attached to the cyclitol ring.