An alternative method for the certification of the sulfur mass fraction in coal Standard Reference Materials

The S mass fractions of coal SRMs 2682b, 2684b, and 2685b are certified by direct comparison with coal SRMs 2682a, 2684a, and 2685a, respectively, using high-temperature combustion analysis with infrared (IR) absorption detection. The S mass fractions of the "a" materials used for calibration were previously determined by means of isotope-dilution thermal-ionization mass spectrometry (ID-TIMS). Therefore, the comparisons performed with the combustion-IR absorption method establish direct traceability links to accurate and precise ID-TIMS measurements. The expanded uncertainties associated with the certified S mass fractions are of approximately the same magnitude as would be expected for the ID-TIMS methodology. An important aspect of these certifications is that each "b" material is essentially identical with the corresponding "a" material, because both were produced from the same bulk, homogenized coal. As a test of the efficacy of the new certification approach when calibrant and unknown are not identical, the S mass fraction of coal SRM 2683b has been determined by direct comparison to coal SRM 2683a. These two coals, which have both previously been analyzed with ID-TIMS, are different in terms of S content and other properties. Whereas the S mass fraction for SRM 2683b determined with the new methodology agrees statistically with the ID-TIMS value, there is reason for caution in such cases. In addition to the usefulness of the alternative approach for certification activities within NIST, this approach might also be an excellent way of establishing NIST traceability during the value assignment process for reference materials not issued by NIST. Further research is needed, however, to understand better the scope of applicability.     

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.     

Synthesis and antiinflammatory activity of the sulfoxides of 4-[3-(dimethylamino)propyl] - 3,4- dihydro- 2-(1-hydroxyethyl)-3-phenyl-2H-1,4-benzothiazine

Oxidation of 4-[3-(dimethylamino)propyl]-3,4-dihydro-2-(1-hydroxyethyl)-3-phenyl-2H-1,4-benzothiazine, hydrochloride (I) with hydrogen peroxide yielded a mixture of two sulfoxides (II). Since this mixture exhibited antiinflammatory activity, the two components (Isomers A and B) were prepared in purified form by oxidation of I with N-chlorosuccinimide and hydrogen peroxide, respectively. Isomer A was more potent than Isomer B in the carrageenin-induced edema test.     

Studies on phosphatidylserine by tandem quadrupole and multiple stage quadrupole ion-trap mass spectrometry with electrospray ionization: Structural characterization and the fragmentation processes

Low-energy CAD product-ion spectra of various molecular species of phosphatidylserine (PS) in the forms of [M−H]⁻ and [M−2H+Alk]⁻ in the negative-ion mode, as well as in the forms of [M+H]⁺, [M+Alk]⁺, [M−H+2Alk]⁺, and [M−2H+3Alk]⁺ (where Alk=Li, Na) in the positive-ion mode contain rich fragment ions that are applicable for structural determination. Following CAD, the [M−H]⁻ ion of PS undergoes dissociation to eliminate the serine moiety (loss of C₃H₅NO₂) to give a [M−H−87]⁻ ion, which equals to the [M−H]⁻ ion of a phoshatidic acid (PA) and give rise to a MS³-spectrum that is identical to the MS²-spectrum of PA. The major fragmentation process for the [M−2H+Alk]⁻ ion of PS arises from primary loss of 87 to give rise to a [M−2H+Alk−87]⁻ ion, followed by loss of fatty acid substituents as acids (R_xCO₂H, x=1,2) or as alkali salts (e. g., R_xCO₂Li, x=1,2). These fragmentations result in a greater abundance of [M−2H+Alk−87−R₂CO₂H]⁻ than [M−2H+Alk−87−R₁CO₂H]⁻ and a greater abundance of [M−2H+Alk−87−R₂CO₂Li]⁻ than [M−2H+Alk−87−R₁CO₂Li]⁻; while further dissociation of the [M−2H+Alk−87−R_{2(or 1)}CO₂Li]⁻ ions gives a preferential formation of the carboxylate anion at sn-1 (R₁CO₂⁻) over that at sn-2 (R₂CO₂⁻). Other major fragmentation process arises from differential loss of the fatty acid substituents as ketenes (loss of R_x′CH=CO, x=1,2). This results in a more prominent [M−2H+Alk−R₂′CH=CO]⁻ ion than [M−2H+Alk−R₁′CH=CO]⁻ ion. Ions informative for structural characterization of PS are of low abundance in the MS²-spectra of both the [M+H]⁺ and the [M+Alk]⁺ ions, but are abundant in the MS³-spectra. The MS²-spectrum of the [M+Alk]⁺ ion contains a unique ion corresponding to internal loss of a phosphate group probably via the fragmentation processes involving rearrangement steps. The [M−H+2Alk]⁺ ion of PS yields a major [M−H+2Alk−87]⁺ ion, which is equivalent to an alkali adduct ion of a monoalkali salt of PA and gives rise to a greater abundance of [M−H+2Alk−87−R₁CO₂H]⁺ than [M−H+2Alk−87−R₂CO₂H]⁺. Similarly, the [M−2H+3Alk]⁺ ion of PS also yields a prominent [M−2H+3Alk−87]⁺ ion, which undergoes consecutive dissociation processes that involve differential losses of the two fatty acyl substituents. Because all of the above tandem mass spectra contain several sets of ion pairs involving differential losses of the fatty acid substituents as ketenes or as free fatty acids, the identities of the fatty acyl substituents and their positions on the glycerol backbone can be easily assigned by the drastic differences in the abundances of the ions in each pair.     

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.     

Structural Distinction of Diacyl-, Alkylacyl,and Alk-1-Enylacyl Glycerophosphocholines as [M – 15]– Ions by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

We describe a linear ion-trap (LIT) multiple-stage (MSⁿ) mass spectrometric approach towards differentiation of alkylacyl, alk-1-enylacyl- and diacyl-glycerophoscholines (PCs) as the [M – 15]^– ions desorbed by electrospray ionization (ESI) in the negative-ion mode. The MS⁴ mass spectra of the [M – 15 – R₂′CH = CO]^– ions originated from the three PC subfamilies are readily distinguishable, resulting in unambiguous distinction of the lipid classes. This method is applied to two alkyl ether rich PC mixtures isolated from murine bone marrow neutrophils and kidney, respectively, to explore its utility in the characterization of complex PC mixture of biological origin, resulting in the realization of the detailed structures of the PC species, including various classes and many minor isobaric isomers.

Elucidation of the double-bond position of long-chain unsaturated fatty acids by multiple-stage linear ion-trap mass spectrometry with electrospray ionization

Linear ion-trap (LIT) MS2 mass spectrometric approach toward locating the position of double bond(s) of unsaturated long-chain fatty acids and toward discerning among isomeric unsaturated fatty acids as dilithiated adduct ([M-H+2Li]+) ions are described in this report. Upon resonance excitation in a LIT instrument, charge-remote fragmentation that involves beta-cleavage with gamma-H shift (McLafferty rearrangement) is the predominant fragmentation pathway seen for the [M-H+2Li]+ ions of monoenoic long-chain fatty acids. The fragmentation process results in a dilithiated product ion of terminally unsaturated fatty acid, which undergoes consecutive McLafferty rearrangement to eliminate a propylene residue, and gives rise to another dilithiated adduct ion of terminally unsaturated fatty acid. In addition to the above-cited fragmentation process, the [M-H+2Li]+ ions of homoconjugated dienoic long-chain fatty acids also undergo alpha-cleavage(s) with shift of the allylic hydrogen situated between the homoconjugated double bonds to the unsaturated site. These fragmentation pathways lead to two types of CC bond cleavages that are allylic (alpha-cleavage) or vinylic, respectively, to the proximal CC double bond, resulting in two distinct sets of ion series, in which each ion series is separated by a CH2CHCH (40 Da) residue. These latter fragmentations are the predominant processes seen for the polyunsaturated long-chain fatty acids. The spectrum feature dependent on the position of unsaturated double bond(s) affords unambiguous assignment of the position of double bond(s) of long-chain unsaturated fatty acids.     

Characterization of inositol phosphorylceramides from <Emphasis Type="Italic">Leishmania major</Emphasis> by tandem mass spectrometry with electrospray ionization

We describe tandem mass spectrometric approaches, including multiple stage ion-trap and source collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization (ESI) to characterize inositol phosphorylceramide (IPC) species seen as [M - H](-) and [M - 2H + Li](-) ions in the negative-ion mode as well as [M + H](+), [M + Li](+), and [M - H + 2Li](+) ions in the positive-ion mode. Following CAD in an ion-trap or a triple-stage quadrupole instrument, the [M - H](-) ions of IPC yielded fragment ions reflecting only the inositol and the fatty acyl substituent of the molecule. In contrast, the mass spectra from MS(3) of [M - H - Inositol](-) ions contained abundant ions that are readily applicable for assignment of the fatty acid and long-chain base (LCB) moieties. Both the product-ion spectra from MS(2) and MS(3) of the [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) ions also contained rich fragment ions informative for unambiguous assignment of the fatty acyl substituent and the LCB. However, the sensitivity of the ions observed in the forms of [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) (Alk = Li, Na) is nearly 10 times less than that observed in the [M - H](-) form. In addition to the major fragmentation pathways leading to elimination of the inositol or inositol monophosphate moiety, several structurally informative ions resulting from rearrangement processes were observed. The fragmentation processes are similar to those previously reported for ceramides. While the tandem mass spectrometric approach using MS(n) (n = 2, 3) permits the structures of the Leishmania major IPCs consisting of two isomeric structures to be unveiled in detail, tandem mass spectra from constant neutral loss scans may provide a simple method for detecting IPC in mixtures.     

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.     

Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: A mechanistic study

Structural characterization of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PI-4P), and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) by collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization is described. In negative ion mode, the major fragmentation pathways under low energy CAD for PI arise from neutral loss of free fatty acid substituents ([M - H - RxCO2H]-) and neutral loss of the corresponding ketenes ([M - H - R'xCH=C=O]-), followed by consecutive loss of the inositol head group. The intensities of the ions arising from neutral loss of the sn-2 substituent as a free fatty acid ([M - H - R2CO2H]-) or as a ketene ([M - H - R'2CH=C=O] ) are greater than those of ions reflecting corresponding losses of the sn-1 substutient. This is consistent with our recent finding that ions reflecting those losses arise from charge-driven processes that occur preferentially at the sn-2 position. These features permit assignment of the position of the fatty acid substituents on the glycerol backbone. Nucleophilic attack of the anionic phosphate onto the C-1 or the C-2 of the glycerol to which the fatty acids attached expels sn-1 (R1CO2-) or sn-2 (R2CO2-) carboxylate anion, respectively. This pathway is sterically more favorable at sn-2 than at sn-1. However, further dissociations of [M - H - RxCO2H - inositol] , [M - H - RxCO2H]-, and [M - H - RxCH=C=O]- precursor ions also yield RxCO2- ions, whose abundance are affected by the collision energy applied. Therefore, relative intensities of the RxCO2- ions in the spectrum do not reflect their positions on the glycerol backbone and determination of their regiospecificities based on their ion intensities is not reliable. The spectra also contain specific ions at m/z 315, 279, 259, 241, and 223, reflecting the inositol head group. The last three ions are also observed in the tandem spectra of the [M - H]- ions of phosphatidylinositol monophosphate (PI-P) and phosphatidylinositol bisphosphate (PI-P2), in addition to the ions at m/z 321 and 303, reflecting the doubly phosphorylated inositol ions. The PI-P2 also contains unique ions at m/z 401 and 383 that reflect the triply phosphorylated inositol ions. The [M - H]- ions of PI-P and PI-P2 undergo fragmentation pathways similar to that of PI upon CAD. However, the doubly charged ([M - 2H]2-) molecular ions undergo fragmentation pathways that are typical of the [M - H]- ions of glycerophosphoethanolamine, which are basic. These results suggest that the further deprotonated gaseous [M - 2H]2 ions of PI-P and PI-P2 are basic precursors.