Individual Positional Isomers Research Articles

Differentiating Positional Isomers of Nucleoside Modifications by Higher-Energy Collisional Dissociation Mass Spectrometry (HCD MS).

The analytical identification of positional isomers (e.g., 3-, N4-, 5-methylcytidine) within the > 160 different post-transcriptional modifications found in RNA can be challenging. Conventional liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approaches rely on chromatographic separation for accurate identification because the collision-induced dissociation (CID) mass spectra of these isomers nearly exclusively yield identical nucleobase ions (BH2+) from the same molecular ion (MH+). Here, we have explored higher-energy collisional dissociation (HCD) as an alternative fragmentation technique to generate more informative product ions that can be used to differentiate positional isomers. LC-MS/MS of modified nucleosides characterized using HCD led to the creation of structure- and HCD energy-specific fragmentation patterns that generated unique fingerprints, which can be used to identify individual positional isomers even when they cannot be separated chromatographically. While particularly useful for identifying positional isomers, the fingerprinting capabilities enabled by HCD also offer the potential to generate HPLC-independent spectral libraries for the rapid analysis of modified ribonucleosides. Graphical Abstract ᅟ.

Exploiting charge differences for the analysis of challenging post-translational modifications by capillary electrophoresis-mass spectrometry

Reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with mass spectrometry (MS) is typically employed for mapping modifications in proteins and peptides. Here we applied a low-flow capillary electrophoresis (CE) −electrospray ionization interface coupled to Orbitrap mass spectrometers to analyze challenging modifications such as asparagine deamidation, aspartate isomerization, arginine citrullination, and phosphopeptide isomers. We achieved excellent resolution of asparagine (Asn), aspartic acid (Asp) and isoaspartic acid (iso-Asp) containing peptides using a synthetic peptide mixture. The migration order in CE enabled a clear assignment of in vitro deamidation/isomerization sites in a protein standard mixture of intermediate complexity (48 proteins) as well as the determination of the in vivo deamidation rate of histone H1.0 directly in a crude nuclear protein fraction. Besides these well-known modifications citrullination, a post-translational modification which changes the positively charged guanidinium group of arginine to the uncharged ureido group of citrulline, was investigated. Applying CE-MS for fast and sensitive analyses of various post-translational modifications of intact and enzymatically digested histone H4, we were able to detect a variety of citrullinated proteoforms. MS/MS analysis with electron transfer dissociation (ETD) fragmentation identified the presence of deiminated Arg at position 3 and 17 of histone H4. Moreover, based on CE-MS, isobaric mono-phosphorylated peptides obtained in the course of a kinase activity study were separated and individual positional isomers quantified.

How big is big? Separation by conventional methods, X-ray and electronic structures of positional isomers of bis-tert-butylisocyano adduct of 2(3),9(10),16(17),23(24)-tetrachloro-3(2),10(9),17(16),24(23)-tetra(2,6-di-iso-propylphenoxy)-phthalocyaninato iron(II) complex

A presence of bulky 2,6-di-iso-propylphenoxy groups in bis-tert-butylisocyano adduct of 2(3),9(10),16(17),23(24)-tetrachloro-3(2),10(9),17(16),24(23)-tetra(2,6-di-iso-propylphenoxy)-phthalocyaninato iron(II) complex allows separation of two individual positional isomers and a mixture of the remaining two isomers using conventional chromatography. X-ray structures of “[Formula: see text]” and “[Formula: see text]” isomers were confimed by X-ray crystallography. Density functional theory (DFT) and time-dependent DFT (TDDFT) calculations of each individual positional isomer allowed insight into their electronic structures and vertical excitation energies, which were correlated with the experimental UV-vis and MCD spectra.

Trans-octadecenoic Acid Positional Isomers Have Different Accumulation and Catabolism Properties in Mice.

Trans fatty acids (TFA) are considered risk factors for cardiovascular disease (CVD), while the details of distribution and metabolism of the individual isomers are not clear. Here we investigated the accumulation and catabolic rate of TFA positional isomers of octadecenoic acid (18:1) in mice. ICR mice were fed deuterium- and [1-(13)C] stable isotope-labeled trans-9-18:1 (9t-18:1*), trans-10-18:1 (10t-18:1*), or trans-11-18:1 (11t-18:1*) for 2 or 4 weeks, or a TFA mixture (9t-18:1*, 10t-18:1*, and 11t-18:1*) for 3 weeks. Analysis of whole-body tissues by gas chromatography-chemical ionization mass spectrometry revealed the highest 9t-18:1* levels in the heart. Significant differences in the accumulation of the respective trans-18:1 were observed in the heart and erythrocytes, where 9t- > 11t- > 10t-18:1*, but no significant difference was observed in the liver or white adipose tissue (WAT). Mice fed on 11t-18:1 demonstrated accumulation of endogenously synthesized conjugated linoleic acid in the liver, WAT, and heart, but any other metabolites were not found in other groups. Furthermore, we analyzed catabolic rates of single-dose-administered trans-18:1* isomers into [(13)C]-labeled CO2 using isotope-ratio mass spectrometry, and the 10t-18:1*catabolic rate was significantly higher than those of 9t- and 11t-18:1*. We found that the accumulation and catabolism of trans-18:1 positional isomers varied in these mice. Differential accumulation in tissues suggests that individual TFA positional isomers may play different roles in human health.

Coupling ferrocene to brominated tetraazaporphyrin: exploring an alternative synthetic pathway for preparation of ferrocene-containing tetraazaporphyrins

A Castro--Stephens coupling reaction between metal-free 3(2),8(7)-dibromo- 2(3),7(8),12(13),17(18)-tetra-tert-butyl-5,10,15,20-tetraazaporphyrin and (ferrocenylethynyl)copper resulted in the formation of copper 2(3),7(8),12(13), 17(18)-tetra-tert-butyl-3(2), 8(7)-di(ferrocenylethynyl)-5,10,15,20-tetraazaporphyrin and copper 2(3),7(8),12(13),17(18)-tetra-tert-butyl-3(2)-ferrocenylethynyl-5, 10,15,20-tetraazaporphyrin, which were separated in the form of 2 positional isomers along with copper 3(2)-bromo-2(3),7(8),12(13),17(18)-tetra-tert-butyl-5,10,15,20-tetraazaporphyrin and copper 2(3),7(8),12(13),17(18)-tetra-tert-butyl-5,10,15,20-tetraazaporphyrin. A similar reaction with metal-free 3(2),8(7),13(12), 18(17)-tetrabromo-2(3),7(8),12(13),17(18)-tetra-tert-butyl-5,10,15,20-tetraazaporphyrin resulted in only a trace amount of 3(2),8(7),13(12)-tribromo-2(3),7(8),12(13),17(18)-tetra-tert-butyl-18(17) -ferrocenylethynyl-5,10,15,20-tetraazaporphyrin, while no products with larger number of organometallic substituents were observed. Direct coupling between ferrocenelithium and 3(2),8(7)-dibromo-2(3),7(8),12(13),17(18)-tetra-tert-butyl-5,10,15,20-tetraazaporphyrin resulted in a debromination reaction accompanied by very minor dimerization of the tetraazaporphyrin core, which was explained based on the steric properties of the parent tetraazaporphyrin. The target compounds were characterized using APCI mass spectrometry, UV-vis, and MCD spectroscopy, while the electronic structure of ferrocenylethyl-containing products was predicted by DFT approach. X-ray structures of individual positional isomers of copper 2-bromo-3,7,12,18-tetra-tert-butyl-5,10,15,20-tetraazaporphyrin and 3, 7, 12,18-tetrabromo-2,8,13,17-tetra-tert-butyl-5,10,15,20-tetraazaporphyrin were also discussed.

Modeling of the energies and splitting of the Qx and Qy bands in positional isomers of zinc pyridinoporphyrazines by TDDFT approach: Can TDDFT help distinguishing the structural isomers?

Electronic structures, energies and splitting of the Qx and Qy bands for positional isomers of zinc mono-, di-, tri-, and tetra pyridinoporphyrazines as well as parent zinc phthalocyanine were investigated using density functional theory (DFT) and time-dependent (TD) DFT approaches. The influence of the Hartree–Fock exchange on excited state energies and Qx and Qy bands splitting were studied using GGA BP86 and hybrid B3LYP and PBE1PBE exchange-correlation functionals. Solvent effects were estimated using the polarized continuum model (PCM) approach and cyclohexane, toluene, or DMSO as solvents. It was found that general trends in the Qx and Qy band energies and splitting correlate very well with the available experimental data on pyridinoporphyrazines and follow the trends in HOMO–LUMO and HOMO–LUMO+1 energy gaps as well as LUMO–LUMO+1 splitting. TDDFT trends allow estimation of the Qx and Qy band energies and splitting in unknown tripyridinoporphyrazines and in individual positional isomers of tetrapyridinoporphyrazines.

<i>In Vivo</i> Pharmacokinetics and Pharmacodynamics of Positional Isomers of Mono-PEGylated Recombinant Human Granulocyte Colony Stimulating Factor in Rats

In this study, the pharmacokinetic and pharmacodynamic properties of Lys(35), Met(N-terminal), and Lys(17)-mono-PEGylated recombinant human granulocyte colony stimulating factor (rhG-CSF) positional isomers were evaluated in rats. The in vitro biological activities of Lys(35), Met(N-terminal), and Lys(17)-mono-PEGylated rhG-CSF were determined by examining NFS-60 cell proliferation. Plasma concentrations of rhG-CSF and white blood cell (WBC) counts and absolute neutrophil conunt (ANC) were measured and pharmacokinetic and pharmacodynamic properties were determined after a single subcutaneous administration of the Lys(35), Met(N-terminal), or Lys(17) isomers at 0.1 mg/kg in rats. The in vitro biological activities of Lys(35), Met(N-terminal), and Lys(17)-mono-PEGylated rhG-CSF individual positional isomers were 20.1%, 37.4%, and 15.3%, respectively, that of rhG-CSF. However, all three mono-PEGylated rhG-CSF isomers had a greater blood half-life (T1/2) and in vivo efficacy as determined by WBC counts and ANC than rhG-CSF, but no significant difference was observed between the three isomers. In conclusion, Lys(35), Met(N-terminal), and Lys(17)-mono-PEGylated rhG-CSF individual positional isomers exhibit an enhanced the in vivo pharmacokinetics and pharmacodynamics. Furthermore, three isomers have comparable in vivo pharmacokinetic and pharmacodynamic properties, but their in vitro biological activities are PEGylation site dependent.

Structural identification and biological activity of positional isomers of long-acting and mono-PEGylated recombinant human granulocyte colony-stimulating factor with trimeric-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group

The individual positional isomers from the mono-PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were successfully isolated with additional strong cation exchange chromatography using Source 15S. The three isolated individual positional isomers were found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), analytical size exclusion high-performance liquid chromatography (SE-HPLC), and analytical cation exchange HPLC (CIE-HPLC) and were also characterized with respect to site of PEGylation by enzymatic digestion with endoproteinase Lys-C and N-terminal sequencing. In addition, in vitro biological activity was determined by cell proliferation assay. It was determined that the three isolated individual positional isomers were PEGylated at Lys35, MetN-terminal, and Lys17 of the rhG-CSF molecule with a 23-kDa trimer-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group (mPEG-NHS). All individual positional isomers (Lys35-PEGylated rhG-CSF, MetN-terminal-PEGylated rhG-CSF, and Lys17-PEGylated rhG-CSF) retained in vitro biological activity and were found to be 18.5%, 37.6%, and 7.1%, respectively, compared with the rhG-CSF molecule. The significantly different in vitro biological activities observed in the individual positional isomers could be presumably due to interference of receptor binding or active sites on the rhG-CSF molecule. In conclusion, the individual positional isomers isolated from the mono-PEGylated rhG-CSF were well characterized with respect to the site of PEGylation involving Lys35, MetN-terminal, and Lys17. This characterization of the individual positional isomers would be critical to provide a basis for establishing consistency in the manufacturing process.

Use of ionically tagged glycosyl donors in the synthesis of oligosaccharide libraries

We have developed a new procedure based on the random glycosyl reaction of a partially benzoylated glycosyl acceptor with a glycosyl donor containing a 4,6- O-(4-methoxycarbonylbenzylidene) protecting group as a masked/caged ion-tag. Glycosylated products are ionically tagged by saponification of the methyl ester and the use of this anion-tag greatly simplifies the separation of the desired oligosaccharides from unreacted or excess glycosyl acceptors as well as from over-glycosylated oligosaccharides. In addition, the use of partially benzoylated acceptors greatly improves their solubility in dichloromethane increasing the yield of product formation and, also, of altering the distribution of positional isomers in favor of products derived by reaction of the donors at hydroxyl groups which otherwise would be considerably less reactive. Using this new approach in random glycosyl reactions, several oligosaccharide libraries were readily prepared in overall yields of 60–70% and the individual positional isomers present in the libraries were identified using the ‘reductive-cleavage’ method.

Kinetic and J-Resolved Statistical Total Correlation NMR Spectroscopy Approaches to Structural Information Recovery in Complex Reacting Mixtures: Application to Acyl Glucuronide Intramolecular Transacylation Reactions

We demonstrate here a new variant on a statistical spectroscopic method for recovering structural information on unstable intermediates formed in reaction mixtures. We exemplify this approach with respect to the internal acyl migration reactions of 1-beta-O-acyl glucuronides (AGs), which rearrange at neutral or slightly alkaline pH on a minute to hour time scale to yield a series of positional glucuronide ring isomers and alpha/beta anomers from the 1-beta (starting material), i.e. 2-beta, 2-alpha, 1-alpha, 3-beta, 3-alpha, and 4-beta, 4-alpha isomers together with the aglycon and alpha- and beta-glucuronic acid hydrolysis products. Multiple sequential 800 MHz cryoprobe (1)H NMR spectra (1D and 2D J-resolved, JRES) were collected on a 5.1 mM solution of a synthetic model drug glucuronide, 1-beta-O-acyl (S)-alpha-methyl phenylacetyl glucuronide (MPG) in 0.1 M sodium phosphate buffer in D2O at pD 7.4 over 18 h to monitor the reaction which leads to the formation of the eight positional isomers and hydrolysis products. As the reaction proceeds and new isomers form, the NMR signal intensities vary accordingly allowing the application of a novel kinetic variant on statistical total correlation spectroscopy (K-STOCSY) method to recover the connectivities between proton signals on the same reacting molecule based on their intensity covariance through time. We performed K-STOCSY analysis on both the standard 1D NMR spectra and the skyline projected singlets of the (1)H-(1)H JRES NMR spectra through time, i.e. the K-JRES-STOCSY experimental variant, which increases the effective spectral dispersion and is ideally suited for the analysis of heavily overlapped spin systems. High statistical correlations were observed between mutarotated alpha- and beta-anomers of individual positional isomers, as well as directly acyl migrated products and anticorrelation observed between signals from compounds that were being depleted as others increased, e.g. between the 1-beta and 2-alpha/2-beta isomers. This statistical kinetic approach enabled the recovery of structural connectivity information on all isomers allowing unequivocal resonance assignment, and this approach to spectroscopic information recovery has wider potential uses in the study of reactions that occur on the second-to-minute time scale in conditions where multiple sequential NMR spectra can be collected. JRES-STOCSY is also of potential use as a method for recovering spectroscopic information in highly overlapped NMR signals and spin systems in other types of complex mixture analysis.

Structural and Biophysical Characterization of the 40 kDa PEG−Interferon-α2aand Its Individual Positional Isomers

The human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFNalpha(2a). (40)PEG-IFNalpha(2a) is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of (40)PEG-IFNalpha(2a) and its positional isomers, in comparison with IFNalpha(2a), using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFNalpha(2a) is not modified by the presence of the polymer in all positional isomers constituting (40)PEG-IFNalpha(2a). Regardless of where the PEG-polymer is attached, it adopts a very mobile and flexible random coil conformation, producing a shield for the protein without a permanent coverage of the protein surface. Hydrodynamic data indicate that the protein-attached PEG has a slightly more compact random-coil structure than the free PEG-polymer. Our results also provide evidence of significant structural and physicochemical advantages conferred by the pegylation: increase of the effective hydrodynamic volume and modification of the molecular shape, higher temperature stability, and reduced tendency for aggregation. These results are of tremendous pharmacological interest and benefit as was clinically shown with PEGASYS. This study constitutes a new standard for the characterization of pegylated proteins and enables an important step toward the understanding on a molecular level of the binding of (40)PEG-IFNalpha(2a) and its positional isomers to its cellular receptors.

Structural, Kinetic, and Thermodynamic Analysis of the Binding of the 40 kDa PEG−Interferon-α2a and Its Individual Positional Isomers to the Extracellular Domain of the Receptor IFNAR2

Type-I Interferons exert antiviral and antiproliferative activities through the binding to a common cell surface receptor comprising two subunits, IFNAR1 and IFNAR2. Human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) used to treat various cancers and viral diseases including Hepatitis B/C infections. To significantly improve the pharmacological properties of the drug, a pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side-chain of IFNalpha(2a). Here, we report the detailed structural, kinetic, and thermodynamic analysis of the binding to the extracellular domain of the receptor IFNAR2 of (40)PEG-IFNalpha(2a) and its isolated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively, in comparison with unmodified IFNalpha(2a). Our binding studies, using the surface plasmon resonance technique, show that the pegylation does not abolish the binding to the receptor, but significantly reduces the affinity mainly due to a change of the association rate. The results are supported by modeling and simulation of the binding, using Self-Avoiding-Walk calculations for the polymer conformations. A correlation between the structural parameters and the kinetic and thermodynamic parameters of the binding of the positional isomers could be established. For the Isomer-K31 and -K164, the PEG-polymer attachment point is located in proximity to the binding interface, and the isomers display affinity in the range 150-520 nM in an enthalpy-driven binding process. In contrast for the Isomer-K134, -K131, -K121, and -K70, the PEG-polymer is attached remotely from the binding interface, and the isomers exhibit a higher affinity (32-76 nM) in an entropy-driven binding process. This study constitutes an essential collection of knowledge on which the interaction of (40)PEG-IFNalpha(2a) and its positional isomers with its cellular receptors can be better understood.

CMC and regulatory considerations for polyethylene glycol-modified protein therapeutics

The hepatitis C virus (HCV) is currently estimated to infect over 270 million people worldwide, with up to 4 million people newly infected each year. It is the leading cause of chronic liver disease and the most common reason for liver transplantation in the USA and Europe. The pegylated interferons (peginterferon alfa-2b: PEG-INTRON® and peginterferon alfa-2a: PEGASYS®), alone or in combination with ribavirin, are the current standard of care for patients with HCV. There are significant chemistry and manufacturing control (CMC) challenges in the manufacture and characterization of the pegylated interferons since these therapeutic drugs are composed of heterogeneous mixtures of mono-pegylated proteins with differing sites of attachment of polyethylene glycol (PEG) to the core interferon molecule, termed “positional isomers”. These CMC issues were comprehensively addressed during the development of PEG-INTRON®. Extensive characterization of the pegylated interferon in the drug substance and drug product, and the establishment of appropriate manufacturing controls, served as the foundation for a successful CMC strategy to obtain regulatory approval. This study describes the analytical strategies for quantitating the positional isomer populations which comprise PEG-INTRON®. Individual positional isomers were also isolated and characterized with respect to site of pegylation and in vitro biological potency. It was determined that different pegylation chemistries result in different mixtures of positional isomers. PEG-INTRON® is predominantly pegylated at histidine 34, demonstrated to be the most biologically active positional isomer of the pegylated interferons. In contrast, the antiviral activities of positional isomers generated by attachment of PEG to lysine residues were significantly lower. Thus, the differences in pegylation chemistry between PEG-INTRON® and PEGASYS® result in different distributions of pegylated positional isomers and differential biological activity profiles in vitro.

Characterization of cigarette smoke condensates by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOFMS). Part 2: basic fraction.

Cigarette smoke condensate is a complex chemical matrix. Analysis of nitrogen-containing compounds present therein is very difficult because of the limitation of the peak capacity of conventional one-dimensional chromatography. Extensive and laborious sample preparation is frequently required or selective detectors are frequently used. In this study, the basic fraction of mainstream cigarette smoke condensate has been investigated by using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOFMS). Auto data processing by TOFMS software combined with manual identification was used to assign the components. 377 nitrogen-containing compounds, including 155 pyridine derivatives, 104 quinoline/isoquinoine derivatives, and 56 pyrazine derivatives were tentatively identified. By selecting appropriate unique masses and in the light of the component positions in the structured chromatogram, alkyl-substituted pyridines, pyrazines, and quinolines/isoquinolines were separately shown and further validated. The peaks of eight individual positional isomers of two-carbon-substituted pyridines and thirteen positional isomers of methyl-substituted quinolines/isoquinolines were further confirmed, based on linear incremental retention behavior in combination with TOFMS and the structured chromatogram of GC x GC.

Silver-ion high-performance liquid chromatographic separation and identification of conjugated linoleic acid isomers.

This is the first report of the application of silver-ion impregnated high-performance liquid chromatography (Ag(+)-HPLC) to the separation of complex mixtures of conjugated linolenic acid (CLA) isomers present in commercial CLA sources and foods and in biological specimens. This method showed a clear separation of CLA isomers into three groups related to their trans,trans, cis,trans or trans,cis, and cic,cis configuration of the conjugated double-bond system. In addition, this method separated individual positional isomers of the conjugated diene system within each geometrical isomeric group. Following Ag(+)-HPLC isolation, gas chromatography (GC)-electron impact mass spectrometry, and GC-direct deposition-Fourier transformed infrared spectroscopy were used to confirm the identity of two major positional isomers in the cis/trans region, i.e., delta 8,10- and delta 11,13-octadecadienoic acids, which had not been chromatographically resolved previously. Furthermore, the potential of this method was demonstrated by showing different Ag(+)-HPLC profiles exhibiting patterns of isomeric distributions for biological specimens from animals fed a diet containing a commercial CLA preparation, as well as for a commercial cheese product.

13C nuclear magnetic resonance spectral confirmation of Δ6‐and Δ7‐trans‐18:1 fatty acid methyl ester positional isomers

AbstractTrans octadecenoic acid methyl ester isomers were obtained from a partially hydrognated soybean oil and isolated by silver‐ion high‐performance liquid chromatography. Recently, the double‐bond positions for nine individual trans octadecenoic acid positional isomers (Δ8 through Δ16) were confirmed by gas chromatography‐electron ionization mass spectrometry after derivatization to 2‐alkenyl‐4,4‐dimethyloxazoline. In this communication, the presence of two additional trans‐18:1 fatty acid methyl ester positional isomers (Δ6 and Δ7) in the same mixture is confirmed by 13C nuclear magnetic resonance spectroscopy. The identity of the Δ5‐trans‐18:1 fatty acid methyl ester positional isomer is inferred.

Quantitative Gas Chromatography–Mass Spectrometry Isomer-Specific Measurement of Hydroxy Fatty Acids in Biological Samples and Food as a Marker of Lipid Peroxidation

We have developed a capillary gas chromatography–mass spectrometry method for the quantitative analysis of individual positional isomers of monohydroxy fatty acids derived from linoleic, arachidonic, eicosapentaenoic, or docosahexaenoic acid. Peroxidation of a particular polyunsaturated fatty acid results already in a complex mixture of positional isomers of hydroperoxy and hydroxy fatty acids. Catalytic hydrogenation of lipid extracts produces stable saturated hydroxy lipids from the complex mixtures typical of oxidized biological samples, simultaneously simplifying the analytical problem and eliminating oxidation artifacts. After saponification and methylation, monohydroxy fatty acid methyl esters are purified by solid-phase extraction and partially resolved using a CP Sil 19 column following on-column derivatization of the hydroxy groups with tetramethylammonium hydroxide. The resulting methoxy fatty acid methyl esters are subjected to electron impact mass spectroscopy. Two characteristic ions are produced for each positional isomer. Quantitative measurements were achieved by using odd chain C17 and C19 monohydroxy fatty acids as internal standards. The limit of detection of individual hydroxy fatty acid isomers is dependent on the total number of ions monitored. Monitoring 11 pairs of ions simultaneously gives limits of detection of 10 ng. Sensitivity is much higher by monitoring fewer ions and as little as 0.2 ng of a single isomer can be detected. The method has been applied for the quantitative analysis of hydroxy (plus hydroperoxy) fatty acids in plasma, adipose tissue, oils, and foods. To date over 1000 samples have been analyzed using the method described in this paper.

Spectral confirmation of trans monounsaturated C18 fatty acid positional isomers

AbstractThe trans octadecenoic acid methyl ester isomers were obtained from a partially hydrogenated soybean oil sample and isolated by silver‐ion high‐performance liquid chromatography. The double bond configuration was confirmed to be trans by using gas chromatography‐direct deposition‐Fourier transform infrared spectroscopy. The double bond positions for nine individual trans octadecenoic acid positional isomers were confirmed by gas chromatography‐electron ionization mass spectrometry after derivatization to 2‐alkenyl‐4,4‐dimethyloxazoline. These nine trans positional isomers were resolved on either one of the two polar 100% cyanopropylpolysiloxane 100‐m capillary columns, SP 2560 and Cp‐Sil 88, at an isothermal temperature of 140°C. These nine isomers were confirmed to have double bonds at carbons C‐8 through C‐16. The pair of trans octadecenoic acid positional isomers with double bonds at C‐13 and C‐14 are reported for the first time to be resolved by gas chromatography.

750 MHz HPLC−NMR Spectroscopic Studies on the Separation and Characterization of the Positional Isomers of the Glucuronides of 6,11-Dihydro-11- oxodibenz[b,e]oxepin-2-acetic Acid

Ester glucuronides (beta-1-O-acyl-D-glucopyranuronates) of many drugs can undergo a series of acyl migration reactions, resulting in positional isomers and anomers which can react with serum proteins with possible toxicological consequences. We have investigated the acyl migration of the ester glucuronides of the model drug 6,-11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid in pH 7.4 buffer using directly coupled 750 MHz stopped-flow HPLC-NMR spectroscopy. Using a reversed phase isocratic HPLC method with 21% acetonitrile and 79% D2O in the mobile phase, it was possible to separate and hence identify the individual positional isomers of the model drug glucuronide by 750 MHz HPLC-NMR. The order of elution of the isomers from the C18 column was 4alpha-, 4beta-, aglycon, 1beta-, 3beta-, 3alpha-, 2alpha-, 2beta- (alpha- and beta- referring to the anomerization state at C1 on the glucuronide ring and the numbers referring to the carbon number on the glucuronide ring to which the drug moiety has migrated). It is shown that directly coupled ultra-high-field HPLC-NMR spectroscopy offers a unique analytical advantage for obtaining structural information of interconverting compounds in equilibrium mixtures, and this method will be of value in the study of reactive drug glucuronides of toxicological importance.

Identification of isomeric hydroxy fatty acids in aerosol samples by capillary gas chromatography—mass spectrometry

Isomeric mixtures of 5- to (ω — 1)-monohydroxy fatty acids have been isolated from remote marine aerosols by methanolic potassium hydroxide extraction and silica gel column chromatography. Structures of the hydroxy acids were determined by comparison with high-resolution gas chromatography-mass spectrometry (GC—MS) of authentic standards, employing methyl esters and methyl ester trimethylsilyl (TMS) derivatives. A series of monohydroxy acid positional isomers have been identified in the range C 7C 15 with 5-hydroxyundecanoic and 10-hydroxyundecanoic acids as major isomers. Electron-impact mass spectra of hydroxy acid methyl esters show characteristic α-cleavage ions ( e.g. m/z 131 for the 5-hydroxy homologue) and their methanol-eliminated ions ( e.g. m/z 99). Mass spectra of the TMS derivatives also yield characteristic ions at m/z 133 + 14 m ( m ⩾ 5; m = position of carbon atom bearing the hydroxyl group). These specific ions provide a useful tool in mass chromatography to detect individual positional isomers of hydroxy fatty acids in environmental samples.