Structural determination of glycosphingolipids as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisional-activated dissociation on a triple stage quadrupole instrument

Structural characterization of glycosphingolipids as their lithiated adducts using low-energy collisional-activated dissociation (CAD) tandem mass spectrometry with electrospray ionization (ESI) is described. The tandem mass spectra contain abundant fragment ions reflecting the long chain base (LCB), fatty acid, and the sugar constituent of the molecule and permit unequivocal identification of cerebrosides, di-, trihexosyl ceramides and globosides. The major fragmentation pathways arise from loss of the sugar moiety to yield a lithiated ceramide ion, which undergoes further fragmentation to form multiple fragment ions that confirm the structures of the fatty acid and LCB. The mechanisms for the ion formation and the possible configuration of the fragment ions, resulting from CAD of the lithiated molecular ions ([M + Li]+) of monoglycosylceramides are proposed. The mechanisms were supported by CAD and source CAD tandem mass spectra of various cerebrosides and of their analogous molecules prepared by H-D exchange. Constant neutral loss and precursor ion scannings to identify galactosylceramides with sphingosine or sphinganine LCB subclasses, and with specific N-2-hydroxyl fatty acid subclass in mixtures are also demonstrated.     

Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization

We applied electrospray ionization (ESI) tandem quadrupole mass spectrometry to establish the fragmentation pathways of ceramides under low energy collisional-activated dissociation (CAD) by studying more than thirty compounds in nine subclasses. The product-ion spectra of the [M + Li]+ ions of ceramides contain abundant fragment ions that identify the fatty acyl substituent and the long-chain base (LCB) of the molecules, and thus, the structure of ceramides can be easily determined. Fragment ions specific to each ceramide subclasses are also observed. These feature ions permit differentiation among different ceramide subclasses. The ion series arising from the classical C-C bond cleavages that were reported in the fast-atom bombardment (FAB)-high energy tandem mass spectrometry is not observable; however, the product-ion spectra contain multiple fragment ions informative for structural characterization and isomer identification. We also investigated the tandem mass spectra of the fragment ions generated by in-source CAD (pseudo-MS3) and of the deuterium-labeling molecular species obtained by H/D exchange to support the ion structure assignments and the proposed fragmentation pathways that lead to the ion formation.     

Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry

Electrospray ionization (ESI) tandem mass spectrometry (MS) has simplified analysis of phospholipid mixtures, and, in negative ion mode, permits structural identification of picomole amounts of phospholipid species. Collisionally activated dissociation (CAD) of phospholipid anions yields negative ion tandem mass spectra that contain fragment ions representing the fatty acid substituents as carboxylate anions. Glycerophosphocholine (GPC) lipids contain a quaternary nitrogen moiety and more readily form cationic adducts than anionic species, and positive ion tandem mass spectra of protonated GPC species contain no abundant ions that identify fatty acid substituents. We report here that lithiated adducts of GPC species are readily formed by adding lithium hydroxide to the solution in which phospholipid mixtures are infused into the ESI source. CAD of [MLi⁺] ions of GPC species yields tandem mass spectra that contain prominent ions representing losses of the fatty acid substituents. These ions and their relative abundances can be used to assign the identities and positions of the fatty acid substituents of GPC species. Tandem mass spectrometric scans monitoring neutral losses of the head-group or of fatty acid substituents from lithiated adducts can be used to identify GPC species in tissue phospholipid mixtures. Similar scans monitoring parents of specific product ions can also be used to identify the fatty acid substituents of GPC species, and this facilitates identification of distinct isobaric contributors to ions observed in the ESI/MS total ion current.     

Studies on sulfatides by quadrupole ion-trap mass spectrometry with electrospray ionization: Structural characterization and the fragmentation processes that include an unusual internal galactose residue loss and the classical charge-remote fragmentation

The structural characterization of sulfatides by collisional-activated dissociation (CAD) quadrupole ion-trap tandem mass spectrometric methods with electrospray ionization is described. When subjected to CAD in the negative-ion mode, the [M - H]- ions of sulfatides yield abundant structurally informative ions that permit unequivocal assignments of the long-chain base, and fatty acid constituent including the location of double bond. The identification of the position of the double bond on the fatty acyl substituent is based on the observation of the series of the ions arising from classical charge-remote fragmentation processes similar to those observed by high-energy CAD and by tandem quadrupole mass spectrometry. An unusual internal galactose residue loss due to a rearrangement process was also observed. The [M - H]- ions of sulfatides also dissociates to a ceramide anion, which undergoes consecutive fragmentation processes to yield ions informative for identification of the ceramide moiety and permits distinction the sulfatide with a sphingosine subclass from that with a sphinganine long-chain base subclass. The MS(2)-spectra of the sulfatide subclass with a sphingosine LCB and a alpha-hydroxy fatty acyl substituent (d18:1/hFA-sulfatide) are featured by the prominent ion sets of m/z 568, 550, 540, and 522, originated from a primary cleavage of the fatty acyl CO-CH(OH) bond, and are readily differentiable from those arising from the non-hydroxy sulfatide subclass (d18:1/nFA-sulfatide), in which the ion sets are of low abundance. The fragmentation pathways of sulfatides under low-energy CAD are proposed. The pathways are supported by the MS(2)- and MS(3)-spectra of various compounds, and of their H-D exchanged analogs.     

Distinction among isomeric unsaturated fatty acids as lithiated adducts by electrospray ionization mass spectrometry using low energy collisionally activated dissociation on a triple stage quadrupole instrument

Features of tandem mass spectra of dilithiated adduct ions of unsaturated fatty acids obtained by electrospray ionization mass spectrometry with low-energy collisionally activated dissociation (CAD) on a triple stage quadrupole instrument are described. These spectra distinguish among isomeric unsaturated fatty acids and permit assignment of double-bond location. Informative fragment ions reflect cleavage of bonds remote from the charge site on the dilithiated carboxylate moiety. The spectra contain radical cations reflecting cleavage of bonds between the first and second and between the second and third carbon atoms in the fatty acid chain. These ions are followed by a closed-shell ion series with members separated by 14 m/z units that reflect cleavage of bonds between the third and fourth and then between subsequent adjacent pairs of carbon atoms. This ion series terminates at the member reflecting cleavage of the carbon-carbon single bond vinylic to the first carbon-carbon double bond. Ions reflecting cleavages of bonds distal to the double bond are rarely observed for monounsaturated fatty acids and are not abundant when they occur. For polyunsaturated fatty acids that contain double bonds separated by a single methylene group, ions reflecting cleavage of carbon-carbon single bonds between double bonds are abundant, but ions reflecting cleavages distal to the final double bond are not. Cleavages between double bonds observed in these spectra can be rationalized by a scheme involving a six-membered transition state and subsequent rearrangement of a bis-allylic hydrogen atom to yield a terminally unsaturated charge-carrying fragment and elimination of a neutral alkene. The location of the beta-hydroxy-alkene moiety in ricinoleic acid can be demonstrated by similar methods. These observations offer the opportunity for laboratories that have tandem quadrupole instruments but do not have instruments with high energy CAD capabilities to assign double bond location in unsaturated free fatty acids by mass spectrometric methods without derivatization.     

Structural characterization of triacylglycerols as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisionally activated dissociation on a triple stage quadrupole instrument

We describe features of tandem mass spectra of lithiated adducts of triacylglycerol (TAG) species obtained by electrospray ionization mass spectrometry (ms) with low-energy collisionally activated dissociation (CAD) on a triple stage quadrupole instrument. The spectra distinguish isomeric triacylglycerol species and permit assignment of the mass of each fatty acid substituent and positions on the glycerol backbone to which substituents are esterified. Source CAD-MS2 experiments permit assignment of double bond locations in polyunsaturated fatty acid substituents. The ESI/MS/MS spectra contain [M + Li - (RnCO2H)]+, [M + Li - (RnCO2Li)]+, and RnCO+ ions, among others, that permit assignment of the masses of fatty acid substituents. Relative abundances of these ions reflect positions on the glycerol backbone to which substituents are esterified. The tandem spectra also contain ions reflecting combined elimination of two adjacent fatty acid residues, one of which is eliminated as a free fatty acid and the other as an alpha, beta-unsaturated fatty acid. Such combined losses always involve the sn-2 substituent, and this feature provides a robust means to identify that substituent. Fragment ions reflecting combined losses of both sn-1 and sn-3 substituents without loss of the sn-2 substituent are not observed. Schemes are proposed to rationalize formation of major fragment ions in tandem mass spectra of lithiated TAG that are supported by studies with deuterium-labeled TAG and by source CAD-MS2 experiments. These schemes involve initial elimination of a free fatty acid in concert with a hydrogen atom abstracted from the alpha-methylene group of an adjacent fatty acid, followed by formation of a cyclic intermediate that decomposes to yield other characteristic fragment ions. Determination of double bond location in polyunsaturated fatty acid substituents of TAG is achieved by source CAD experiments in which dilithiated adducts of fatty acid substituents are produced in the ion source and subjected to CAD in the collision cell. Product ions are analyzed in the final quadrupole to yield information on double bond location.     

Structural determination of sphingomyelin by tandem mass spectrometry with electrospray ionization

Alkaline metal adduct ions of sphingomyelin were formed by electrospray ionization in positive ion mode. Under low energy collisionally activated dissociation (CAD), the product ion spectra yield abundant fragment ions representative of both long chain base and fatty acid which permit unequivocal determination of the structure. Tandem spectra obtained by constant neutral loss scanning permit identification of sphingomyelin class and specific long chain base subclass in the mixture. The fragmentation pathways under CAD were proposed, and were further confirmed by source CAD tandem mass spectrometry. The total analysis of sphingomyelin mixtures from bovine brain, bovine erythrocytes, and chicken egg yolk is also presented.     

Electrospray ionization/tandem quadrupole mass spectrometric studies on phosphatidylcholines: the fragmentation processes

We applied low-energy collisionally activated dissociation (CAD) tandem quadrupole mass spectrometry to study the fragmentation pathways of the [M + H](+) and [M + Li](+) ions of phosphatidylcholine (PC), generated by electrospray ionization (ESI). It is revealed that the fragmentation pathways leading to loss of the polar head group and of the fatty acid substituents do not involve the hydrogens attached to the glycerol backbone as previously reported. The pathway for formation of the major ion of m/z 184 by loss of the polar head group from the [M + H](+) precursor of a diacyl PC involves the participation of the alpha-hydrogen of the fatty acyl substituents, whereas the H(+) participates in the loss of fatty acid moieties. The alpha-hydrogens of the fatty acid substituents also participate in the major fragmentation processes, including formation of [M + Li-R(x)CO(2)H](+) and [M + Li-59-R(x)CO(2)H](+) ions for the [M + Li](+) ions of diacyl PCs, when subjected to low-energy CAD. These fragmentation processes are deterred by substitution of the fatty acyl moieties with alkyl, alkenyl, or hydroxyl groups and consequentially, result in a distinct product-ion spectrum for various PC, including diacyl-, plasmanyl- plasmenyl-, and lyso-PC isomers. The alpha-hydrogens of the fatty acyl substituents at sn-2 are more labile than those at sn-1. This is reflected by the preferential loss of the R(1)CO(2)H over the R(2)CO(2)H observed for the [M + Li](+) ions of diacyl PCs. The spectrum features resulting from the preferential losses permit identification and assignment of the fatty acid moieties in the glycerol backbone. The new fragmentation pathways established by tandem and source CAD tandem mass spectra of various PC molecules, including deuterium-labeling analogs, were proposed. These pathways would clarify the mechanisms underlying the ion formations that lead to the structural characterization of PC molecules.     

Metal ion complexes in the structural analysis of phospholipids by electrospray ionization tandem mass spectrometry

The effects of metal cationization on collisionally activated dissociation (CAD) of phospholipids were investigated by electrospray ionization with quadrupole ion trap tandem mass spectrometry. The metal ions include Li(+), Na(+), K(+), Sr(2+), Ba(2+), and the first transition series. CAD of the transition metal ion-bound lipid complexes gave significant yields of product ions that identify the positions of the two fatty acyl substituents on the glycerophospholipid backbone. The cobalt(II) ion, which has a single naturally occurring isotope, was expected to be a better cationization reagent as it produces simpler mass spectra than other transition metal ions. CAD of the cobalt(II) ion complexes of glycerophosphoethanolamines, glycerophosphoglycerols and glycerophosphoserines yielded product ions that revealed information regarding both the lipid classes and the regiospecific positions of the two fatty acyl substituents.     

Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization

Negative-ion electrospray ionization tandem quadrupole mass spectrometry provides a useful method for the structural characterization of ceramides. Fragment ions referring to the identities of the fatty acid substituent and of the long chain base of the molecules are readily available and the structure of ceramides can be easily determined. A unique fragmentation pathway which leads to formation of the fatty acid carboxylate anions (RCO2) was observed. This fragmentation is initiated by cleavage of the C2-C3 bond of the LCB to yield a N-acylaminoethanol anion ([RCONHCH2CH2O]-), followed by rearrangement to a carboxyethylamine ([RCO2CH2CH2NH]-) intermediate, which further dissociates to a RCO2- ion. This pathway is confirmed by the CAD tandem mass spectrum of the synthetic N-acylaminoethanol standard and of the deuterated analogs of ceramides obtained by H-D exchange. The observation of RCO2- ion species permits an unambiguous identification of the fatty acyl moiety of ceramides. Tandem mass spectrometry methods for characterization of structural isomers of ceramides using product-ion scanning and for identification of specific ceramide subclasses in biological mixtures using neutral loss scanning are also demonstrated.     

Doubly charged porphyrin ion tandem mass spectrometry: Implications for structure elucidation

Under electron ionization (EI) conditions, porphyrins yield unusually high intensities of doubly charged molecular and fragment ions. These doubly charged ions offer unique opportunities for the structure elucidation of porphyrins by tandem mass spectrometry (MS/MS). First, they fragment to a greater extent than the corresponding singly charged ions under both EI/MS and EI/MS/MS conditions. Second, doubly and singly charged porphyrin ions often fragment via different pathways, and can therefore yield different structural information. This paper describes several ways in which analyses of doubly charged porphyrin ions with a triple quadrupole tandem mass spectrometer can be useful in structure elucidation of porphyrins. The effect of the metal atom on the fragmentation of metalloporphyrins in an EI source is demonstrated by correlating the extent of doubly charged fragment ion formation to a stability index. Doubly charged porphyrin ions are shown to yield predominantly doubly charged daughter ions upon collisionally activated dissociation (CAD), and are also shown to fragment to a greater extent than corresponding singly charged porphyrin ions. Advantages and disadvantages of doubly charged porphyrin ion MS/MS for structure elucidation are discussed.     

A novel structural analysis of glycerophosphocholines as TFA/K(+) adducts by electrospray ionization ion trap tandem mass spectrometry

When collisionally activated dissociation (CAD) of glycerophosphocholine (GPC) species is examined using quadrupole ion trap mass spectrometry (QITMS), the spectral patterns differ from those obtained using sector or quadrupole mass spectrometry. Methods employed in the structural analysis of GPCs using a sector or quadrupole mass spectrometers are not necessarily useful for an ion trap mass spectrometer. A novel method is presented for structurally analyzing GPCs that involves the CAD of trifluoroacetic acid (TFA) adducts of kaliated GPCs. Solutions of GPCs in 0.1% TFA/methanol were electrosprayed to produce precursor ions by attaching a trifluoroacetic acid (TFA) molecule to a kaliated GPC molecule. The CAD-MS/MS spectra obtained by QITMS revealed a dramatic increase in the abundance of fragment ions, corresponding to the losses of sn-1 and sn-2 fatty acyl substituents. A preferential loss of the sn-1 fatty acyl group over the loss of the sn-2 fatty acyl group was observed among the GPC standards examined. A GPC extract from egg yolk was directly analyzed by this method without prior separation. The identities and positions of fatty acyl substituents of over 20 GPC species were identified. Some isomers present in very low relative abundance, which could not be analyzed by QITMS/MS using other ions as precursors, were identified by the TFA attachment method.     

Charge-driven fragmentation processes in diacyl glycerophosphatidic acids upon low-energy collisional activation. A mechanistic proposal

A mechanistic study of diacyl glycerophosphatidic acid (GPA) under low energy collisionally activated decomposition (CAD) with electrospray ionization tandem mass spectrometry is reported. The fragmentation pathways leading to the formation of carboxylate anions [RxCO2-], (x = 1, 2) and the formation of the ions representing neutral loss of fatty acid ([M-H-RxCO2H] ) and neutral loss of ketene ([M-H-R'xCH-C=O] ) (Rx=R'xCH2) are charge-driven processes that are governed by the gas-phase basicity and the steric configuration of the molecules. The preferential formation of the ions of [M-H-R2CO2H]- > [M-H-R1CO2H]- and [M-H-R'2CH=C=O]- > [M-H-R'1CH=C=O]- are attributed to the fact that loss of fatty acid and loss of ketene are sterically more favorable at sn-2. While the observation of the abundance of [M-H-RxCO2H]- > [M-H-R'xCH=C=O]- is attributed to the acidity of the gas phase ion of GPA, which undergoes a more facile neutral loss of acid than loss of ketene. The major pathway leading to the formation of RxCO2- ion under low energy CAD arises from further fragmentation of the [M-H-RxCO2H]- ions by neutral loss of 136, resulting in an abundance of R1CO2- > R2CO2-. The differential formation of the carboxylate anions permits accurate assignment of the regiospecificity of the fatty acid substituents of GPA molecules by tandem mass spectrometry.     

Structural characterization of cardiolipin by tandem quadrupole and multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization

We report negative-ion electrospray tandem mass spectrometric methods for structural characterization of cardiolipin (CL), a four-acyl-chain phospholipid containing two distinct phosphatidyl moieties, of which structural assignment of the fatty acid residues attached to the glycerol backbones performed by low-energy CAD tandem mass spectrometry has not been previously described. The low-energy MS2-spectra of the [M - H]- and [M - 2H]2- ions obtained with ion-trap or with tandem quadrupole instrument combined with ion-trap MS3-spectra or with source CAD product-ion spectra provide complete structural information for CL characterization. The MS2-spectra of the [M - H]- ions contain two sets of prominent fragment ions that comprise a phosphatidic acid, a dehydrated phosphatidylglycerol, and a (phosphatidic acid + 136) anion. The substantial differences in the abundances of the two distinct phosphatidic anions observed in the MS2-spectra of the [M -H]- ions lead to the assignment of the phosphatidyl moieties attached to the 1' or 3' position of central glycerol. Upon further collisional dissociation, the MS3-spectra of the phosphatidic anions provide information to identify the fatty acyl substituents and their position in the glycerol backbone. The MS2-spectra of the [M - 2H]2- ions obtained with TSQ or ITMS contain complementary information to confirm structural assignment. The applications of the above methods in the differentiation of cardiolipin isomers and in the identification of complex cardiolipin species consisting of multiple molecular structures are also demonstrated.     

Charge-remote fragmentation during FAB-CAD-B/E linked-scan mass spectrometry of aminoethyl-triphenylphosphonium derivatives of fatty acids

The carboxyl group of fatty acids is derivatized by aminoethyl triphenylphosphonium (AETPP) bromide. Fast atom bombardment (FAB) collision-activated dissociation (CAD) B/E linked-scan mass spectrometry of these fixed-charge derivatives shows typical charge-remote fragmentation (CRF). Locations of various structural modifications in fatty acids can be recognized easily from CAD spectra of the AETPP derivatives. Because the triphenylphosphonium group localizes positive charge in the molecule, and because a key requirement for CRF is a tightly localized charge site, these preionized molecules fragment under FAB-CAD conditions more effectively than other derivatives that involve ionic bonding with metal cations or protonation of basic sites. Thus, CAD of AETPP derivatives is likely to produce more structurally informative spectra and provide an opportunity to gain additional under-standing of the CRF process. The most profound difference between the AETPP derivatives and other cations in positive mode FAB-CAD-B/E-MS is reflected in the substantial improvement of detection limits for the AETPP derivatives over those for the metal cation adducts. For several fatty acids (C₁₀-C₂₂) tested, the detectability can be enhanced by one to two orders of magnitude when the analysis is performed on the AETPP derivative. In addition, for the analysis of fatty acid mixtures, the FAB mass spectrum of AETPP derivatives produces a relative intensity of the molecular ion peak for each component of the mixture that more closely represents its mole fraction than does that of metal ion adducts.     

Characterization of alkylacyl,alk-1-enylacyl and lyso subclasses of glycerophosphocholine by tandem quadrupole mass spectrometry with electrospray ionization

Positive ion tandem quadrupole mass spectrometric methods for structural characterization of the subclasses of sn-glycero-3-phosphocholine (PC), including alkylacyl- and alk-1-enylacylphosphocholine and lysophosphatidylcholine (LPC), are described. Following collisionally activated dissociation, the [M + Li](+) ions generated by electrospray ionization yield abundant informative fragment ions that permit structural determination, and distinction of regioisomers among lysophosphatidylcholine can be easily achieved. In contrast, structurally informative ions arising from [M + H](+) or [M + Na](+) ions are less prominent. The most abundant ion observed in the product-ion spectra of the [M + Li](+) ions of plasmenyl- and plasmanyl-PC and of LPC arises from loss of N(CH(3))(3) ([M + Li - 59](+)). This feature permits their distinction from a product-ion spectrum arising from a diacylphosphatidylcholine, in which the [M + Li - 183](+) ion reflecting loss of phosphocholine is the most prominent. Examples for identification of various subclasses of PC in biological extracts by tandem mass spectrometry applying various constant neutral loss scannings are also shown.     

Examining the collision-induced decomposition spectra of ammoniated triglycerides as a function of fatty acid chain length and degree of unsaturation. I. The OXO/YOY series

A series of positionally pure triglycerides (TAGs) of the form OXO and YOY, where O is the oleate moiety and X and Y are large arrays of different fatty acid moieties, was synthesized and analyzed by reversed-phase high-performance liquid chromatography/tandem mass spectrometry. The intensities of the collision-induced decomposition (CID) products of ammoniated TAGs (ammonium ion adducts) were examined as a function of chain length, degree of unsaturation, double-bond position, and cis/trans configuration of X and Y. The major CID products, the diglyceride fragment ions and the MH+ ion, were plotted as functions of chain length for the saturated and mono-unsaturated series of X and Y. Different trends for each of these series were observed. Trends in the abundances of these fragment ions were also characterized as a function of degree of unsaturation in the TAGs. In general, the fractional abundances of the MH+ ions vary linearly with degree of unsaturation. However, the presence of double bonds positioned close to the carbonyl carbon of the fatty acid chain promotes the formation of the diglyceride fragment ion corresponding to loss of that fatty acid. Mechanisms of the formation and decomposition of ammoniated TAGs are proposed that fit the trends observed in the data. Extensions of this work are described, and a vision of a derived library of CID spectra is discussed as a platform for comprehensive analysis of complex TAG mixtures.     

Characteristic fragmentation behavior of phosphoamino acid conjugates with 3'-azido-3'-deoxythymidine by electrospray ionization tandem mass spectrometry

The conjugates of phosphoamino acids with 3'-azido-3'-deoxythymidine were synthesized and their structures were determined by various spectral methods. In positive and negative ion electrospray mass spectrometry (ESI-MS), the fragmentation pathways were investigated in conjunction with tandem mass spectrometry (MS/MS). The results showed that there were very different characteristic fragment ions in the positive ion MS/MS spectra and the negative ion MS/MS spectra.     

Electrospray ionization mass spectrometry of AZT H-phosphonates conjugated with steroids

AZT H-phosphonates conjugated with steroids were synthesized and determined by positive and negative ion electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). The fragmentation pathways were investigated in detail. There are very different characteristic fragment ions in the positive and negative ion MS/MS spectra. The azide group of compounds 6a and 6b underwent either elimination of HN(3) or rearrangement to an amine in both positive and negative ion mass spectrometry.     

Electrospray ionization mass spectra of amino acid phosphoramidates of adenosine

Amino acid phosphoramidates of adenosine were synthesized and determined by positive and negative ion electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). The fragmentation pathways were investigated. In the positive ion mass spectra abundant characteristic fragment ions appeared, and many complementary ions were found. In the negative ion mass spectra only a few fragment ions were observed, and most of them contained phosphoryl groups. The results show that ESI-MS is a useful tool for structural determination of amino acid phosphoramidates of nucleosides.