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PubMed2005

Conformation of gramicidin a in water: inference from analysis of hydrogen/deuterium exchange behavior by matrix assisted laser desorption ionization mass spectrometry.

Gramicidin A (the major component of gramicidin D) is a highly hydrophobic peptide with very little solubility in water. Hence, the conformation of this peptide has been extensively investigated in organic solvents and model membranes, but not in water. The peptide adopts a beta6.3-helical conformation in the monomeric and dimeric forms. We have investigated the conformation of gramicidin A in water by monitoring hydrogen-deuterium exchange by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Our results indicate that gramicidin A is monomeric and exists in a highly folded conformation. The metal ion bound forms are clearly discernible in the monomers. The presence of the dimeric form is not observed. It is unlikely this is due to the operating conditions or the method used, as both hetero- and homodimers in gramicidin D are detected when methanol is used as a solvent. The present study also establishes that the linear gramicidins retain a history of solvent environment when ions are generated by matrix-assisted laser desorption ionization and analyzed by time-of-flight.

Anti-Infective Agents↗
PubMed2002

Determination of lamivudine/stavudine/efavirenz in human serum using liquid chromatography/electrospray tandem mass spectrometry with ionization polarity switch.

A high-performance liquid chromatography/tandem mass spectrometry (LC-MS-MS) method with ionization polarity switch was developed and validated in human serum for the determination of a lamivudine (3TC)/stavudine (d4T)/efavirenz combination HIV therapy. Solid phase extraction (SPE) was used to extract these anti-HIV drugs and internal standard aprobarbital. A gradient mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer with pH adjusted to 4.5 using glacial acetic acid was utilized to separate these drugs on a hexylsilane column (150 x 2.0 mm i.d.). The total run time between injections was 18 min. The precursor and major product ions of these drugs were monitored on a triple quadrupole mass spectrometer in the multiple reactions monitoring (MRM) mode. Ionization polarity was switched in the middle of the LC run allowing these anti-HIV drugs with different physicochemical properties to be detected simultaneously. The effect of ion suppression from human serum was studied and no interference with the analysis was noted. The method was validated over the range of 1.1-540 ng/mL for 3TC, 12.5-6228 ng/mL for d4T and 1.0-519 ng/mL for efavirenz. The method was shown to be accurate, with intra-day and inter-day accuracy less than 14.0% and precise, with intra-day and inter-day precision less than 13.1%. The extraction recoveries of all analytes were higher than 90%.

Alkynes↗
PubMed2006

Liquid chromatography with electrospray ionization mass spectrometry method for the assay of glucosamine sulfate in human plasma: validation and application to a pharmacokinetic study.

A liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed and validated for the assay of glucosamine sulfate in human plasma. Plasma proteins were precipitated by acetonitrile, followed by vortex mixing and centrifugation. The supernatant was transferred and derivatized with phenyl iso-thiocyanate in acetonitrile at 60 degrees C for 40 min. Chromatographic separation was performed on a C(18) column (Inertsil ODS-3 150 x 2.1 mm i.d., 5 microm, JP) with a mobile phase gradient consisting of 0.2% acetic acid (aqueous) and methanol at a flow-rate of 0.3 mL/min. MS detection using electrospray ionization (ESI) as an interface was used in single ion monitoring mode to determine positive ions at m/z 297. This method was shown to be selective and sensitive for glucosamine sulfate. The limit of detection was 35 ng/mL for glucosamine sulfate in plasma and the linear range was 0.1-20 microg/mL in plasma with a correlation coefficient (r) of 0.9991. The relative standard deviations (RSDs) of intra-day and inter-day assays were 8.7-11.4 and 9.8-12.6%, respectively. Extraction recoveries of glucosamine sulfate in plasma were greater than 73%. This method proved to be simple, reproducible and feasible for pharmacokinetic studies of glucosamine sulfate in healthy volunteers after a single oral administration (1500 mg). The pharmacokinetic parameters and relative bioavailabilities were investigated for both domestic glucosamine sulfate tablet and capsule preparations compared with an imported capsule product.

Area Under Curve↗
PubMed2005

Determination of enantiomeric composition by negative-ion electrospray ionization-mass spectrometry using deprotonated N-(3,5-dinitrobenzoyl)amino acids as chiral selectors.

The ability to use mixtures of deprotonated N-(3,5-dinitrobenzoyl)amino acids as chiral selectors for the determination of enantiomeric composition by electrospray ionization-mass spectrometry is demonstrated. For each experiment, two N-(3,5-dinitrobenzoyl)amino acids were chosen such that each would have opposite selectivity for the enantiomers of the analyte. Electrospray ionization-mass spectrometry, monitored in the negative ion mode, of solutions containing the two N-(3,5-dinitrobenzoyl)amino acids, sodium hydroxide, and the analyte, in a one-to-one mixture of methanol and water, afford peaks in the mass spectrum that correspond to the deprotonated 1:1 analyte-selector complexes. The ratio of the intensities of the complexes in the mass spectrum can be related to the enantiomeric composition of the analyte. Additionally, the sense and extent of chiral recognition is consistent with chromatographic observations, using chiral stationary phases derived from N-(3,5-dinitrobenzoyl)amino acids. Each analysis of enantiomeric composition requires less than 10 s to complete, indicating that this method has great potential for the development of fast-/high-throughput chiral analyses.

Amino Acids↗
PubMed1994

Ionizing radiation induces apoptotic cell death in human TcR-gamma/delta+ T and natural killer cells without detectable p53 protein.

The p53 tumor suppressor gene has been shown to be involved in programmed cell death, apoptosis, in murine immature thymocytes after treatment with ionizing radiation. Ionizing radiation also induces apoptosis in peripheral mature lymphocytes. In this work, we investigated the p53 participation in radiation-induced apoptosis in human peripheral blood lymphocytes (PBL) subpopulations. Exposure to gamma-irradiation resulted in an appreciable induction of apoptotic cell death in TcR-alpha/beta+ (CD4+ and CD8+) T cells, TcR-gamma/delta+ T cells, B cells and natural killer (NK) cells, as assessed by DNA fragmentation as well as the morphological characteristics. Importantly, it was found that there was a marked difference among PBL subpopulations as regards the induction of p53 protein by gamma-irradiation. Similar to previous observations for murine thymocytes, p53 induction in TcR-alpha/beta+ T cells and B cells after gamma-irradiation was evident by Western blot analysis. Radiation-induced apoptosis in TcR-alpha/beta+ T cells and B cells was efficiently inhibited by cycloheximide, indicating the requirement of de novo protein synthesis, including p53 protein, for radiation-induced apoptosis in both subpopulations. In marked contrast, no identifiable levels of p53 protein were induced in either TcR-gamma/delta+ T or NK cells after gamma-irradiation. In addition, it was demonstrated that radiation-induced cell death in TcR-gamma/delta+ T and NK cells could be prevented by interleukin-2, but not by cycloheximide. These results imply that radiation-induced lymphocytic apoptosis can be mediated by p53-dependent or -independent mechanisms.

Adult↗
PubMed2004

Feasibility of nonvolatile buffers in capillary electrophoresis-electrospray ionization-mass spectrometry of proteins.

The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5-10 microM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.

Alkaline Phosphatase↗
PubMed2004

On-line sheathless capillary electrophoresis/nanoelectrospray ionization-tandem mass spectrometry for the analysis of glycosaminoglycan oligosaccharides.

A novel approach in glycosaminoglycomics, based on sheathless on-line capillary electrophoresis/nanoelectrospray ionization-quadrupole time of flight-mass spectrometry (CE/nanoESI-QTOF-MS) and tandem MS of extended chondroitin sulfate/dermatan (CS/DS) oligosaccharide chains is described. The methodology required the construction of a new sheathless CE/nanoESI-QTOF-MS configuration, its implementation and optimization for the high sensitivity analysis of CS/DS oligosaccharide mixtures from conditioned culture medium of decorin transfected human embryonic kidney (HEK) 293 cells. Under newly established sheathless on-line CE/(-)nanoESI conditions for glycosaminoglycan (GAG) ionization and MS detection, single CS/DS oligosaccharide components of extended chain length and increased sulfation degree were identified. Molecular ions corresponding to species carrying 5 and 6 negative charges could be generated for large GAG oligosaccharide species in the negative ion nanoESI-MS. The optimized on-line conditions enabled the detection of molecular ions assigned to oversulfated tetradeca-, octadeca-, and eicosasaccharide CS/DS molecules, which represent the category of largest sulfated GAG-derived oligosaccharides evidenced by CE/ESI-MS. By on-line CE/ESI tandem MS in data-dependent acquisition mode the oversulfated eicosasaccharide species could be sequenced and the localization of the additional sulfate group along the chain could be determined.

Cell Line↗
PubMed2005

Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis-electrospray ionization-quadrupole time of flight-mass spectrometry.

A capillary zone electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometric method was developed for the characterization of oligonucleotides after synthesis, using model compounds. The major difficulty is the adduction of metal cations to the polyanionic backbone of the oligonucleotide sample, resulting in complex spectra and decreased sensitivity. Several approaches were investigated to circumvent this problem. Separation was performed in an ammonium carbonate buffer. During separation, the interfering metal ions were exchanged for ammonium ions, which are less tightly bound to the oligonucleotide when ionized. The influence of the addition of piperidine and imidazole or trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) to the running buffer for further reduction of cation adduction was investigated. Addition of CDTA to the buffer system resulted in a deconvoluted spectrum with very little adducts. On-line sample stacking proved vital to preconcentrate the samples. The pH and the concentration of the ammonium carbonate buffer as well as the electrophoresis voltage were optimized to achieve the best signal response for the oligonucleotides and a maximum reduction of the cation adducts as well as a short analysis time. Finally, the sheath liquid composition was examined for further improvement of the signal. The developed method was used to analyze different oligonucleotides (5000-9200 Da) in light of its use as a final quality control method for oligonucleotides in terms of purity and sequence homogeneity of the synthesized products. In all cases, very little adducts were observed in the deconvoluted spectra, and the relative errors of the measured molecular masses ranged from 3 to 35 ppm.

Buffers↗
PubMed2005

In vitro studies of the genotoxicity of ionizing radiation in human G(0) T lymphocytes.

In an effort to mimic human in vivo exposures to ionizing irradiation, G(0) phase T lymphocytes from human peripheral blood samples were utilized for in vitro studies of the genotoxic effects of (137)Cs low-LET irradiation and (222)Rn high-LET irradiation. Both types of radiation induced mutations in the HPRT gene in a dose-dependent manner, with a mutant frequency (MF) = 4.28 + 1.34x + 7.51x(2) for (137)Cs (R(2) = 0.95) and MF = 4.81 + 0.67x for (222)Rn (R(2) = 0.51). Post (137)Cs irradiation incubation in the presence of cytosine arabinoside, a reversible inhibitor of DNA repair, caused an increase in the MF over irradiation alone, consistent with a misrepair mechanism being involved in the mutagenicity of low-LET irradiation. The spectrum of (137)Cs irradiation-induced mutation displayed an increase in macro-deletions (in particular total gene deletions) and rearrangement events, some of which were further defined by either chromosome painting or direct DNA sequencing. The spectrum of (222)Rn irradiation-induced mutation was characterized by an increase in small alterations, especially multiple single base deletions/substitutions and micro-deletions. These studies define the specific response of human peripheral blood T cells to ionizing irradiation in vitro and form a basis for evaluating the genotoxic effects of human in vivo exposure.

Cell Division↗
PubMed2005

Complex chromosome aberrations persist in individuals many years after occupational exposure to densely ionizing radiation: an mFISH study.

Long-lived, sensitive, and specific biomarkers of particular mutagenic agents are much sought after and potentially have broad applications in the fields of cancer biology, epidemiology, and prevention. Many clastogens induce a spectrum of chromosome aberrations, and some of them can be exploited as biomarkers of exposure. Densely ionizing radiation, for example, alpha particle radiation (from radon or plutonium) and neutron radiation, preferentially induces complex chromosome aberrations, which can be detected by the 24-color multifluor fluorescence in situ hybridization (mFISH) technique. We report the detection and quantification of stable complex chromosome aberrations in lymphocytes of healthy former nuclear-weapons workers, who were exposed many years ago to plutonium, gamma rays, or both, at the Mayak weapons complex in Russia. We analyzed peripheral-blood lymphocytes from these individuals for the presence of persistent complex chromosome aberrations. A significantly elevated frequency of complex chromosome translocations was detected in the highly exposed plutonium workers but not in the group exposed only to high doses of gamma radiation. No such differences were found for simple chromosomal aberrations. The results suggest that stable complex chromosomal translocations represent a long-lived, quantitative, low-background biomarker of densely ionizing radiation for human populations exposed many years ago.

Chromosome Aberrations↗
PubMed2002

Increasing endogenous ceramide using inhibitors of sphingolipid metabolism maximizes ionizing radiation-induced mitochondrial injury and apoptotic cell killing.

To enhance the killing effects of ionizing radiation, we amplified the endogenous ceramide signal in Jurkat cell cultures using 3 different inhibitors of sphingolipid metabolism: DL-PDMP, D-MAPP and imipramine. Of the various possible drug combinations, only DL-PDMP (20 microM) + imipramine (20 microM) and DL-PDMP (20 microM) + imipramine (20 microM) + D-MAPP (5 microM) induced a major increase in ceramide levels, reaching 240% and 340% of control values, respectively, after incubation for 48 hr. With these models, we demonstrate that endogenously formed ceramide triggers time- and concentration-dependent apoptosis through induction of mitochondrial injury and activation of the caspase pathway. Cellular dysfunction includes alterations to the cellular redox potential, as assessed by the generation of ROS and total glutathione depletion, and a drop in Delta Psi(m). A parallel elevation of mitochondrial ceramide levels was also observed. The combination of DL-PDMP + imipramine +/- D-MAPP with 10 Gy irradiation produced cumulative effects leading to apoptosis via mitochondrial collapse and activation of the caspase cascade. The association efficiency was confirmed in normal and acid sphingomyelinase-deficient lymphoid cell lines. Taken together, these results suggest that increasing endogenous ceramide levels may potentially be very valuable when combined with ionizing radiation in tumor therapy.

Apoptosis↗
PubMed2003

NF-kappaB activation plays an antiapoptotic role in human leukemic K562 cells exposed to ionizing radiation.

Exposure of cells to ionizing radiation (IR) determines cellular lesions, such as DNA and membrane damage, which involve a coordinate network of signal transduction pathways responsible for resistance to or delay of apoptosis, depending on cell type and administered dose. Since, after IR exposure, the apoptotic profile appeared different in the two chosen cell lines K562 and Jurkat along with caspase-3 activation, we paid attention to the influence exerted by Protein kinase C delta on transcription factor NF-kappaB activation. Interestingly, K562 resist to IR carrying out a survival strategy which includes PKC delta/NF-kappaB pathway activation, probably mediated by novel IKKs and a role for PI-3-kinase in activating PKC delta at Thr 505 by PDK-1 phosphorylation is suggested. In addition, since caspase-3 is not activated in these cells upon ionizing radiation exposure, it could be supposed that NF-kappaB antagonizes apoptosis induction interfering with pathways which lead to caspase activation, may be by inducing expression of IAP, caspases 3, 7, 9, inhibitor. Thus NF-kappaB activation explains the resistance displayed by K562 to IR and drug potential interference directed to this protein could overcome apoptosis resistance in clinical settings.

Apoptosis↗
PubMed2006

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.

Antipsychotic Agents↗
PubMed2001

Analysis of alcohols, as dimethylglycine esters, by electrospray ionization tandem mass spectrometry.

Dimethylglycine (DMG) esters are new derivatives for the rapid, sensitive and selective analysis of primary and secondary alcohols, in complex mixtures, by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Their development was inspired by the use of the complementary dimethylaminoethyl esters for the trace, rapid analysis of fatty acids. DMG esters are simply prepared by heating a dichloromethane solution of the imidazolide of dimethylglycine, containing triethylamine, and an alcohol. DMG esters of long-chain fatty alcohols, isoprenoidal alcohols and hydroxy-acids are analysed by electrospray ionization tandem mass spectrometry with a precursor ion of m/z 104 scan. Diols, glyceryl esters, glyceryl ethers and some sterols are analysed by a neutral loss of 103 Da scan. Trimethylglycine (TMG) ester iodides, prepared by alkylation of DMG esters with methyl iodide, are more sensitive derivatives for molecules containing secondary alcohol groups, such as cholesterol and gibberellic acid. They are analysed by a precursor ion of m/z 118 scan. DMG or TMG derivatives were shown to be at least comparable and sometimes an order of magnitude more sensitive than N-methylpyridyl ether derivatives for ESI-MS/MS analysis of the different classes of alcohols. Applications of these derivatives for the diagnosis of inherited disorders and the analysis of natural products are presented.

Alcohols↗
PubMed2001

Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry.

The potential of electrospray ionization (ESI) mass spectrometry (MS) to detect non-covalent protein complexes has been demonstrated repeatedly. However, questions about correlation of the solution and gas-phase structures of these complexes still produce vigorous scientific discussion. Here, we demonstrate the evaluation of the gas-phase binding of non-covalent protein complexes formed between bovine pancreatic trypsin inhibitor (BPTI) and its target enzymes over a wide range of dissociation constants. Non-covalent protein complexes were detected by ESI-MS. The abundance of the complex ions in the mass spectra is less than expected from the values of the dissociation constants of the complexes in solution. Collisionally activated dissociation (CAD) tandem mass spectrometry (MS/MS) and a collision model for ion activation were used to evaluate the binding of non-covalent complexes in the gas phase. The internal energy required to induce dissociation was calculated for three collision gases (Ne, Ar, Kr) over a wide range of collision gas pressures and energies using an electrospray ionization source. The order of binding energies of the gas-phase ions for non-covalent protein complexes formed by the ESI source and assessed using CAD-MS/MS appears to differ from that of the solution complexes. The implication is that solution structure of these complexes was not preserved in the gas phase.

Animals↗
PubMed2001

Structural assignment of isomeric 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione mono- and disulfonic acids by liquid chromatography electrospray and atmospheric pressure chemical ionization tandem mass spectrometry.

Positionally isomeric 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione mono- and disulfonic acids give rise to similar electrospray ionization (ESI) and atmosphere pressure chemical ionization (APCI) mass spectra, which show very abundant MH(+) ions and negligible fragmentation. The MH(+) ions of these isomeric acids exhibit notably different behavior under collision-induced dissociation (CID) conditions. The acids with a sulfonic group at position 8' in the quinoline moiety, adjacent to the N-atom, exhibit highly abundant [MH - H(2)SO(3)](+) ions (m/z 272 for the mono- and m/z 352 for the disulfonic acids), which are of lower abundance in the CID spectra of isomers with the SO(3)H group at other positions, remote from the nitrogen atom. The latter isomers undergo efficient eliminations of SO(3) and HSO(3). The isomeric diacids with one SO(3)H group at position 4 of the indene-1,3(2H)-dione moiety, adjacent to one of the carbonyl groups, undergo highly efficient elimination of H(2)O. Mechanistic pathways, involving interactions between adjacent groups, are proposed for the above regiospecific fragmentations. Pronounced different behavior has been also observed in negative ion tandem mass spectrometric measurements of the sulfonic acids. The distinctive behavior of the isomeric acids was strongly pronounced when the measurements were performed with an ion trap mass spectrometer (LCQ), and much less so with a triple-stage quadrupole instrument (TSQ).

Coloring Agents↗
PubMed2001

Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses.

This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.

Electrophoresis↗
PubMed2002

Effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of analytes in liquid chromatographic/ionspray tandem mass spectrometric determination.

The effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of drug molecules in liquid chromatographic/ionspray tandem mass spectrometric (LC/MS/MS) determination were evaluated for simvastatin (SV) and its hydroxy acid (SVA). The objective was to improve further the sensitivity for SV by overcoming the unfavorable condition caused by the formation of multiple major adduct ions and multiple major fragment ions when using ammonium as LC mobile phase buffer. Mobile phases (70:30 acetonitrile-buffer, 2 mM, pH 4.5) with buffers made from ammonium, hydrazine or alkyl (methyl, ethyl, dimethyl or trimethyl)-substituted ammonium acetate were evaluated. Q1 scan and product ion scan spectra were obtained for SV in each of the mobile phases under optimized conditions. The results showed that, with the alkylammonium buffers, the alkylammonium-adducted SV was observed as the only major molecular ion, while the formation of other adduct ions ([M + H](+), [M + Na](+) and [M + K](+)) was successfully suppressed. On the other hand, product ion spectra with a single major fragment ion were not observed for any of the alkylammonium-adducted SVs. The affinity of the alkylammoniums to SV and the basicity of the alkylamines are believed to be factors influencing the formation and abundance of molecular and fragment ions, respectively. Methylammonium acetate provided the most favorable condition among all the buffers evaluated and improved the sensitivity several-fold for SV in LC/MS/MS quantitation compared with that obtained using ammonium acetate buffer. Better precision for SV in both Q1 and SRM scans was observed when using methylammonium buffer compared with those using ammonium buffer. The mobile phase buffer contents did not seem to affect the ionization, fragmentation and chromatography of SVA. The results of this evaluation can be applied to similar situations with other organic molecules in ionspray LC/MS/MS determination.

Anticholesteremic Agents↗