Determination of glycoalkaloids and relative aglycones by nonaqueous capillary electrophoresis coupled with electrospray ionization-ion trap mass spectrometry

Glycoalkaloids are naturally occurring nitrogen-containing compounds present in many species of the family Solanaceae, including cultivated and wild potatoes (Solanum spp.), tomatoes (Lycopersicon spp.), etc. These compounds have pharmacological and toxicological effects on humans due to their significant anticholinesterase activity and disruption of cell membranes. Herein is reported the development of a capillary electrophoresis (CE) method using nonaqueous (NA) separation solutions in combination with ion trap mass spectrometry (MS and MS/MS) detection for the identification and quantification of glycoalkaloids and their relative aglycones. A mixture 90:10 v/v of MeCN-MeOH containing 50 mM ammonium acetate and 1.2 M acetic acid (applied voltage of 25.5 kV) was selected as a good compromise for the separation and detection of these compounds. The electrospray MS measurements were carried out in the positive ionization mode using a coaxial sheath liquid, methanol-water (1:1) with 1% of acetic acid at a flow rate of 2.5 microL/min. Under optimized experimental conditions, the predominant ion was the protonated molecular ion ([M+H](+)) of solanidine (m/z = 398), tomatidine (m/z = 416), chaconine (m/z = 852), solanine (m/z = 868), and tomatine (m/z = 1034). MS/MS experiments were carried out systematically by changing the relative collisional energy and monitoring the intensities of the fragment ions that were not high enough to allow better quantification than with the mother ions. The method was used for analyzing glycoalkaloids in potato extracts.     

Self-assembly formation of the magic ion of (H2O)20O+: observation of nanoscale cages of oxygenated water clusters induced from iron nanoparticles

For the first time, we observed a stable and intense ion (m/z 376) of the oxygenated water cluster ion ((H(2)O)(20)O(+)) produced from simply spraying an aqueous solution of iron nanoparticles (Fe NPs) into an electrospray mass spectrometry (ESI-MS) system. Tandem mass spectrometric (MS/MS and MS/MS/MS) results were applied to identify the assignments of the fragment ions of m/z 376 in order to explore the possible structures of this cluster ion. The possible structures of the (H(2)O)(20)O(+) ions are proposed as pentagonal dodecahedron water clathrate cages from the results of tandem mass spectrometry since eliminations of five water molecules were frequently observed in the MS/MS results for many subsequent fragment ions of m/z 376. The formation of this oxygenated water cluster ion ((H(2)O)(20)O(+)) in ESI-MS is attributed to the high surface reactivity and surface energy of Fe NPs during ESI processes (under high temperature and high voltage (5 kV) of ESI spray environment). We believe that the observation of self-assembly formation of oxygenated water clusters is an important issue in nanoscience as well as in the fields of water clusters.     

Application of nanoscale packed capillary liquid chromatography (75 μm id) and capillary zone electrophoresis/electrospray ionization mass spectrometry to the analysis of macrolide antibiotics

Nanoscale separation techniques, nanoscale packed capillary columns (75 μm id), and capillary zone electrophoresis (CZE), on-line with electrospray mass spectrometry (ESI/MS), were applied to the separation of a series of ten macrolide antibiotics. Both techniques use sub-microliter-per-minute flow rates through the analytical column and therefore require an electrospray probe that incorporates coaxial sheath flow. Positive ion electrospray mass spectra of these compounds yielded mainly protonated molecules. Fragmentation to yield structurally significant fragment ions was achieved by collision-induced dissociation (CID) at increased skimmer voltages. Separations were achieved using both techniques, with CZE/ESI/MS showing improved peak shapes and detection limits combined with faster analysis times. Nanoscale packed capillary columns provided better chromatographic resolution and was less susceptible to peak broadening caused by overloading of the analytes.     

Fragmentation studies on monensin A by sequential electrospray mass spectrometry

Monensin A was studied by electrospray ionisation sequential mass spectrometry (ESI-MSn) and all fragments were confirmed by accurate-mass measurements. Analyses were performed on both a quadrupole time-of-flight (Q-tof) and a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. MSn analysis shows that depending on sample preparation the ion at m/z 671 consists of two different ions with the same accurate-mass. It is either the monensin protonated parent ion or a different ion structure derived from the loss of water from the water adduct of monensin. Both ions show different fragmentation patterns. Major fragment ions from the protonated parent ion were produced by Grob-Wharton type fragmentations in addition to various simple neutral losses. The fragmentation pathways of the two different m/z 671 ions are proposed.     

Determination of rosiglitazone and metformin in human serum by CE-ESI-MS

A new method for the determination of anti-diabetic drugs metformin and rosiglitazone based on the use of capillary electrophoresis with electrospray mass spectrometry was developed. The proposed method allowed their separation within 11 min by using 50 mM formic acid at +20 kV. Positive electrospray ionization and selected ion monitoring [M+H](+) of metformin (m/z=130) and rosiglitazone (m/z=358) were performed. Several important experimental parameters influencing electrospray ionization of metformin and rosiglitazone were studied. The final composition of sheath liquid was water/methanol/formic acid (50:49.5:0.5, v/v/v), at a flow rate of 2 μL/min. The developed method was applied for the determination of metformin and rosiglitazone simultaneously in human serum after protein precipitation with acetonitrile. The limits of detection of developed method were 4.42 and 2.14 ng/mL for rosiglitazone and for metformin, respectively, which is sufficient for therapeutic serum concentration levels monitoring for both studied drugs.     

Identification of diphenhydramine metabolites in human urine by capillary electrophoresis-ion trap-mass spectrometry

The identification of diphenhydramine (DH) metabolites that are frequently observed in the capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) analyses of alkaline liquid/liquid and solid-phase extracts of patient urines is demonstrated. Having standards for DH and diphenhydramine-N-oxide (DHNO), the presence of these two compounds could be confirmed in urines that were collected overnight after administration of 25 mg DH chloride. Using CZE coupled to ion-trap mass spectrometry (CE-MS(n)) with positive electrospray ionization and an acetate buffer at pH 5.6, the [M+H](+) ions of DH (m/z = 256), DHNO (m/z = 272), and nordiphenhydramine (NDH, m/z = 242) and their fragmentation to a common m/z 167 product ion (diphenylcarbinol moiety) was monitored. The data indicate that all three compounds are cations in an acidic environment, the migration order being NDH, DH, and DHNO. Data obtained under negative electrospray ionization conditions suggest the presence of diphenylmethoxyacetic acid-glycine amide ([M-H](-) ion of m/z 298 and fragmentation to m/z 254, loss of CO(2)), a metabolite that could tentatively be assigned to a characteristic peak observed in the MEKC electropherogram at alkaline pH. The data presented in this paper illustrate the value of using CE-MS(n) for identification of urinary drug metabolites for which no standards are available.     

Determination of patulin in apple juice by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

An HPLC-MS-MS method with selected reaction monitoring (SRM) for the determination of patulin in apple juice samples is described. Mass spectrometric detection was accomplished following atmospheric pressure chemical ionization (APCI) in both positive and negative ion modes. Collision induced dissociation (CID) of the protonated molecular ion led initially to the loss of H2O (fragment m/z 137). At higher energies CO is lost from both the protonated parent molecule (fragment m/z 127) and the dehydrated molecular ion (fragment m/z 109). In contrast, CID of the deprotonated molecular ion led initially to the fragment at m/z 109 corresponding to the loss of either CO2 or acetaldehyde, followed at higher CID energy by the loss of H2O (fragment m/z 135) and CO (fragment m/z 125) from the deprotonated molecular ion. Detection in the negative ion mode proved superior and a linear response was observed over the injected range from 6 to 200 ng patulin. Apple juice samples spiked with patulin between 10 and 135 microg/l were analyzed following liquid-liquid extraction with ethyl acetate and clean up with sodium carbonate. Utilizing reversed-phase HPLC with acetonitrile-water (10:90) at 0.5 ml/min, levels down to 10 microg/l were readily quantified and a detection limit of 4 microg/l was attainable at a signal-to-noise (SIN) ratio of 4. The MS data for the spiked samples compared well to the UV data and when plotted against each other displayed a correlation coefficient (R) of 0.99.     

Strategy for structural elucidation of drugs and drug metabolites using (MS)n fragmentation in an electrospray ion trap

Triple-stage quadrupole (TSQ) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and ion trap ESI-MS/MS can be used to cleave protonated molecules to produce carbocations and neutral molecules in the positive ion mode. Dissociation products which correspond to protonated forms of neutral fragment molecules can also be trapped and detected. These protonated molecules in turn can cleave via carbocation cleavage, ipso cleavage, onium cleavage or McLafferty or related rearrangements. One can elucidate the structures of metabolites from the differences in m/z ratios of the fragments arising from the original drug compound and its metabolite. This strategy for structural elucidation is further facilitated by estimates of the reactivity of drugs with oxygen diradicals involved in cytochrome P-450 cycles.     

The identification of in vitro metabolites of CKD-732 by liquid chromatography/tandem mass spectrometry

The structural elucidation of fourteen metabolites of CKD-732, formed in vitro with rat liver microsomes, was performed using high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS). To identify proposed structures of the metabolites, the product ion mass spectra of the protonated molecules ([M + H]+), the retention times on reversed-phase HPLC, and UV-Vis spectra were utilized. Characteristic product ions for the identification of CKD-732 metabolites were observed at m/z 231, 236, and 252. The fragment ions at m/z 236 and 252 indicated the unchanged form and the N-oxide of the dimethylaminoethoxycinnamoyl group, respectively. The ion at m/z 231 indicated the presence of the hydroxylated form of the fumagillol group. The N-oxide of CKD-732, which was detected at m/z 515 and eluted later than CKD-732 in the reversed-phase HPLC system, was measured as a major metabolite. Three cis-trans isomers were also found.     

High-throughput approaches towards the definitive identification of pharmaceutical drug metabolites. 1. Evidence for an ortho effect on the fragmentation of 4-benzenesulfinyl-3-methylphenylamine using electrospray ionisation mass spectrometry

A 50 m/z unit loss from protonated 4-benzenesulfinyl-3-methylphenylamine has been observed and investigated using electrospray ionisation quadrupole ion trap mass spectrometry (ESI-QIT-MS). It was hypothesised that the specific fragmentation was affected by the presence of an ortho methyl group in relation to the sulfoxide functionality, i.e. an ortho effect influences the preferred dissociation pathway. This was because the des-methyl homologue did not display a 50 m/z unit loss. This fragmentation was shown to be a two-step process with sequential losses of a hydroxyl radical and a thiol radical. Molecular modelling calculations showed that the most favourable site of protonation for 4-benzenesulfinyl-3-methylphenylamine was the sulfoxide oxygen, which would facilitate the loss of a hydroxyl radical. Subsequent deuterium-exchange experiments confirmed that the loss was a hydroxyl radical and afforded definitive assignment of the site of protonation. Furthermore, the involvement of a single exchangeable hydrogen atom in the overall 50 m/z unit loss was demonstrated. Thus, supportive evidence was provided for the involvement of the ortho methyl group in the second stage of the fragmentation, leading to the loss of the thiol radical. Accurate mass measurements, performed using electrospray ionisation Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS), verified the elemental formulae of the individual losses. The ion structure following the 50 m/z unit loss was proposed to be a protonated aminofluorene and was supported by comparing the product ion spectrum of commercially available protonated 2-aminofluorene with the MS4 data of protonated 4-benzenesulfinyl-3-methylphenylamine. Fragmentation mechanisms are proposed. The relevance of the loss with regards to pharmaceutical drug metabolite identification is discussed.     

Use of doubly protonated molecules in the analysis of cathedulins in crude extracts of khat (Catha edulis) by liquid chromatography/serial mass spectrometry

Analysis of crude methanolic extracts of fresh khat (Catha edulis) by liquid chromatography/mass spectrometry (LC/MS) revealed the presence of 62 cathedulin alkaloids (compared with 15 published structures). Many cathedulins generated doubly protonated molecules following electrospray ionisation and the ratio of doubly to singly protonated species could be manipulated by adjusting the electrospray capillary position and source conditions. By selecting the doubly protonated species for serial mass spectrometric analysis (MS/MS), it was possible to use an ion trap mass spectrometer to observe singly charged product ions at lower m/z values than ion trap MS/MS analysis of [M+H](+) would have allowed. These spectra were particularly valuable in elucidating the acylation patterns of cathedulins where MS/MS analysis of [M+H](+) resulted in loss of a large neutral species to yield a small singly charged fragment below the lower limit for ion trapping. Acylation patterns for most of the 62 cathedulins are proposed from mass spectrometric analysis, and the data obtained for a major unreported cathedulin of mass 1001 Da suggest that it belongs to a new group of cathedulins having a cathate dilactone bridge but not an evoninate bridge.     

单糖衍生物的电喷雾质谱裂解规律研究

以1-(2-萘基)-3-甲基-5-吡唑啉酮(NMP)作单糖标识剂, 经在线串联的LC-ESI-MS建立了单糖衍生物的电喷雾质谱裂解方法.衍生物在质谱裂解中糖类化合物特有的规范信息.借助糖类化合物在ESI-MS条件下表现出的分子离子峰m/z [M H] , 及在ESI-MS/MS条件下呈现出的特征碎片离子峰m/z 473, 可有效地确定出单糖类化合物的组成. 尽管一些脂肪醛和芳香醛也能同时被标识, 然而在质谱条件下不产生m/z 473的特征碎片离子峰, 且它们的洗脱远在糖类组分之后, 因此不干扰糖类化合物的分离和结构确定.通过建立的LC-ESI-MS方法, 对水解蜂花粉中的单糖进行了分析.结果表明: 水解的蜂花粉中含甘露糖(Man)、半乳糖醛酸(GalUA)、葡萄糖醛酸(GlcUA)、鼠李糖(Rha)、葡萄糖 (Glc)、半乳糖(Gal)、阿拉伯糖(Ara)、木糖(Xyl)和岩藻糖(Fuc).本方法为环境样品中单糖类化合物的确定提供了准确、可靠的技术手段.     

三唑仑苯二氮(艹卓)类药物的电喷雾质谱裂解特征

采用电喷雾/四极杆飞行时间质谱(ESI-QqTOF)联用技术,对3种三唑仑苯二氮(艹卓)类药物进行CID研究,并以质子化准分子离子[M+H]+作为内标物,对碎片离子进行了准确质量测定,确认了这些碎片离子的元素组成,探讨了该类化合物的质谱裂解规律.研究发现,它们的ESI-MS2(源内)和ESI-MS3质谱分别生成脱去N2分子、HCN或CH3CN分子和Cl原子的碎片离子,其中m/z 205为3种药物共有的碎片离子,这些特征可用于三唑仑苯二氮(艹卓)类药物的体内代谢转化和定量研究.     

A simplified procedure for indole alkaloid extraction from Catharanthus roseus combined with a semi-synthetic production process for vinblastine

Dried leaves of Catharanthus roseus were extracted with aqueous acidic 0.1 M solution of HCl. Alkaloid-embonate complexes were obtained as precipitates by treating the extract with an alkaline (NaOH) solution of embonic acid (4,4-methylene-bis-3-hydroxynaphtalenecarboxylic acid). The precipitate mainly consisted of catharanthine and vindoline embonates and it was directly used as the starting material for a semi-synthesis of the anti-cancer bisindole alkaloid vinblastine. The coupling reaction involved oxidation of catharanthine in aqueous acidic medium by singlet oxygen ((1)O2), continuously produced in situ by the reaction between H2O2 with NaClO. An excess of NaBH4 was used for the reduction step. Analysis of the reaction mixture indicated a maximum yield of 20% for vinblastine at pH 8.3, based on the initial amount of catharanthine concentration. Direct-injection electrospray ionization mass spectrometry in positive ion mode was used for the identification of vinblastine. The mass spectra of vinblastine were dominated by the corresponding protonated molecular ion [M+H]+ at m/z 811 and the characteristic fragment ions matched with those of the standard compound.     

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.     

Fragmentation reactions of protonated peptides containing glutamine or glutamic acid

A variety of protonated dipeptides and tripeptides containing glutamic acid or glutamine were prepared by electrospray ionization or by fast atom bombardment ionization and their fragmentation pathways elucidated using metastable ion studies, energy-resolved mass spectrometry and triple-stage mass spectrometry (MS(3)) experiments. Additional mechanistic information was obtained by exchanging the labile hydrogens for deuterium. Protonated H-Gln-Gly-OH fragments by loss of NH(3) and loss of H(2)O in metastable ion fragmentation; under collision-induced dissociation (CID) conditions loss of H-Gly-OH + CO from the [MH - NH(3)](+) ion forms the base peak C(4)H(6)NO(+) (m/z 84). Protonated dipeptides with an alpha-linkage, H-Glu-Xxx-OH, are characterized by elimination of H(2)O and by elimination of H-Xxx-OH plus CO to form the glutamic acid immonium ion of m/z 102. By contrast, protonated dipeptides with a gamma-linkage, H-Glu(Xxx-OH)-OH, do not show elimination of H(2)O or formation of m/z 102 but rather show elimination of NH(3), particularly in metastable ion fragmentation, and elimination of H-Xxx-OH to form m/z 130. Both the alpha- and gamma-dipeptides show formation of [H-Xxx-OH]H(+), with this reaction channel increasing in importance as the proton affinity (PA) of H-Xxx-OH increases. The characteristic loss of H(2)O and formation of m/z 102 are observed for the protonated alpha-tripeptide H-Glu-Gly-Phe-OH whereas the protonated gamma-tripeptide H-Glu(Gly-Gly-OH)-OH shows loss of NH(3) and formation of m/z 130 as observed for dipeptides with the gamma-linkage. Both tripeptides show abundant formation of the y(2)' ion under CID conditions, presumably because a stable anhydride neutral structure can be formed. Under metastable ion conditions protonated dipeptides of structure H-Xxx-Glu-OH show abundant elimination of H(2)O whereas those of structure H-Xxx-Gln-OH show abundant elimination of NH(3). The importance of these reaction channels is much reduced under CID conditions, the major fragmentation mode being cleavage of the amide bond to form either the a(1) ion or the y(1)' ion. Particularly when Xxx = Gly, under CID conditions the initial loss of NH(3) from the glutamine containing dipeptide is followed by elimination of a second NH(3) while the initial loss of H(2)O from the glutamic acid dipeptide is followed by elimination of NH(3). Isotopic labelling shows that predominantly labile hydrogens are lost in both steps. Although both [H-Gly-Glu-Gly-OH]H(+) and [H-Gly-Gln-Gly-OH]H(+) fragment mainly to form b(2) and a(2) ions, the latter also shows elimination of NH(3) plus a glycine residue and formation of protonated glycinamide. Isotopic labelling shows extensive mixing of labile and carbon-bonded hydrogens in the formation of protonated glycinamide.     

Electrospray tandem quadrupole fragmentation of quinolone drugs and related ions. On the reversibility of water loss from protonated molecules

Selected reaction monitoring (SRM) of quinolone drugs showed different sensitivities in aqueous solution vs. biological extract. The authors suggested formation of two singly protonated molecules with different behavior, one undergoing loss of H(2)O and the other loss of CO(2), so that SRM transitions might depend on the ratios of these forms generated by the electrospray. These surprising results prompted us to re-examine several quinolone drugs and some simpler compounds to further elucidate the mechanisms. We find that the relative contributions of loss of H(2)O vs. loss of CO(2) in tandem mass spectrometric (MS/MS) experiments depend not only on molecular structure and collision energy, but also, in certain cases, on the cone voltage. We further find that many product ions formed by loss of H(2)O can reattach a water molecule in the collision cell, whereas ions formed by loss of CO(2) do not. Since reattachment of H(2)O can occur after water loss in the cone region and prior to selection of the precursor ion, this effect leads to the dependence of MS/MS spectra on the cone voltage used in creating the precursor ion, which explains the formerly observed effect on SRM ratios. Our results support the earlier conclusion that varying amounts of two ions of the same m/z value are responsible for problems in the analysis of these drugs, but the origin is in dehydration/rehydration reactions. Thus, SRM transitions for certain complex compounds may be comparable only when monitored under equivalent ion-forming conditions, including the voltage used in the production of the protonated molecules in the electrospray ionization (ESI) source.     

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.     

亲水作用色谱-电喷雾串联质谱法检测原料奶及奶制品中的三聚氰胺

建立了亲水作用色谱-电喷雾串联质谱测定原料奶及奶制品中三聚氰胺的方法。样品采用1%三氯乙酸水溶液-乙腈（体积比为1∶1）混合溶液提取,混合型阳离子交换反相固相萃取柱（MCX）富集净化,亲水作用色谱柱分离,电喷雾串联四极杆质谱仪进行检测。结果表明,三聚氰胺的质量浓度在0.05～10.0 mg/L范围内具有良好的线性关系。原料奶及奶制品中的三聚氰胺在0.5,2.5和10 mg/kg 3个添加水平下,平均回收率为76.3%～98.7%,相对标准偏差均小于6.8%;定量限（S/N＞10）为0.05 mg/kg。     

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).