Extractable organic compounds in polyurethane foam with special reference to aromatic amines and derivatives thereof

Methods for determination of aromatic amines and related compounds in flexible toluene diisocyanate (TDI)-based polyurethane (PUR) foam were investigated. The foam was extracted using 0.1% (w/v) aqueous acetic acid (HAc). Extraction solutions were analysed and aromatic amines were determined as ethyl chloroformate (Et) and pentafluoropropionic acid anhydride (PFPA) derivatives. The determinations were performed using liquid chromatography (LC) and mass spectrometry (MS) detection with electrospray ionisation (ESI) or gas chromatography (GC)-MS with chemical ionisation monitoring negative ions (NCI). The Et derivatives were determined using LC-ESI⁺-MS with detection limit of 2 pg of toluenediamine (TDA). The PFPA derivatives were determined using LC-ESI⁻-MS or GC-NCI-MS with detection limits of 0.1 and 0.02 pg of TDA, respectively. Using trideuterium labelled TDA as internal standard, linear calibration curves were obtained in the range of 0.01-0.50 μg ml⁻¹ (n=7), with correlation coefficients >0.999. When plotting calibration curves for TDA-PFPA derivatives determined using LC-MS against TDA-PFPA using GC-MS and TDA-Et using LC-MS, linear curves were obtained. The relative standard deviation (R.S.D.) for determination of TDA in foam extraction solutions were 13%. LC-MS determination of PFPA derivatives was more selective, as compared to LC-MS of Et derivatives.In foam extraction solutions, 2,4- and 2,6-TDA, several isomers of methylenedianiline (MDA) and dimers of TDA/TDI were observed. 2,4-TDA and 4,4′-MDA are possible human carcinogens. Hydrolysis of the extraction solution revealed a large pool of TDA/TDI compounds and oligomers. The concentration of TDA in foam was affected by the extraction media, temperature and duration. The choice of derivatisation procedure also affected the determination of TDA. In extraction solutions from six different commercially available flexible foam qualities 2,4- and 2,6-TDA were found in the range of 0-7 and 0-6 μg g⁻¹ foam, respectively. When flexible foam was heated, considerable higher concentrations of TDA were observed.     

Determination of aromatic amines in aqueous extracts of polyurethane foam using hydrophilic interaction liquid chromatography and mass spectrometry

A method is presented for the determination of aromatic amines in aqueous extracts of polyurethane (PUR) foam. The method is based on the extraction of PUR foam using aqueous acetic acid (0.1%, w/v) followed by determination of extracted aromatic amines using hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS) with positive electrospray ionisation. The injections of volumes up to 5 μL of aqueous solutions were made possible by on-column focusing with partially filled loop injections. The fragmentation patterns for 2,4- and 2,6-toluene diamine (TDA) and 4,4′-methylene dianiline (MDA) were clarified by performing a hydrogen-deuterium exchange study.TDA and MDA were determined using trideuterated 2,4- and 2,6-TDA and dideuterated 4,4′-MDA as internal standards. Linear calibration graphs were obtained over the range 0.025-0.5 μg mL⁻¹ with correlation coefficients >0.996 and the instrumental detection limit for each compound was <50 fmol. The stability of the amines was influenced by the matrix, so their concentrations decreased over time.Agreement was observed between the results of analyses of PUR foam extracts by HILIC-MS/MS and results obtained by ethyl chloroformate derivatisation and reversed phase (RP) liquid chromatography-mass spectrometry (LC-MS/MS).TDA was observed to be unstable in extracts of foam but not in pure solutions.     

Collision-induced dissociation of corticosteroids in electrospray tandem mass spectrometry and development of a screening method by high performance liquid chromatography/tandem mass spectrometry

A screening method based on liquid chromatography/electrospray tandem mass spectrometry was developed in order to control the illegal use of corticosteroids as growth promoters in cattle. The objective was the detection of low residue levels of corticosteroids or metabolites in biological matrices. Relative to other studies published on this subject, the present work focused on enhancing specificity and sensitivity. Firstly, fragmentation of corticosteroids by collision-induced dissociation was studied. In positive mode, the losses of H(2)O for each hydroxyl group fixed on the molecule, as well as the loss of HF or HCl for halogenated compounds, were observed. For higher collision energy, fragmentations in the B, C and D rings were induced. The negative mode was found to be more specific, inducing a cleavage of the C(20)-C(21) bond with concomitant loss of formaldehyde (CH(2)O). Secondly, three acquisition methods in the negative mode were studied and evaluated, recorded signals being the parent ion [M + acetate](-) and the two daughter ions, [M - H](-) and [M - H - CH(2)O](-). For dexamethasone, MS/MS instrumental detection limits of fragment ion and neutral loss scans, and of multiple reaction monitoring (MRM), were 250, 20 and 5 pg injected, respectively. The MRM method was then evaluated with the objective of use for the detection of corticosteroid residues in biological samples (urine, hair, muscle) and for a metabolism study.     

Solid-phase microextraction liquid chromatography/tandem mass spectrometry for the analysis of chlorophenols in environmental samples

Liquid chromatography with atmospheric pressure chemical ionisation mass spectrometry (LC/APCI-MS), using negative ion detection in a triple quadrupole instrument, was used for the determination of chlorophenols (CPs) in environmental samples. In-source collision-induced dissociation (CID) was compared with MS/MS fragmentation. In general, less fragmentation was observed in MS/MS as compared with in-source CID, with the latter providing more intense fragment ions due to chemical ionisation. Under MS/MS conditions [M - H - HCl](-) was the main fragment ion observed for all compounds except for pentachlorophenol, which showed no fragmentation. For multiple reaction monitoring (MRM) acquisition mode, the transition from [M - H](-) to [M - H - HCl](-) was selected, leading to detection limits down to 0.3 ng injected. Direct and headspace-solid-phase microextraction (HS-SPME) were used as preconcentration procedures for the analysis of CPs in wood and in industrially contaminated soils. CPs were quantified by standard addition, which led to good reproducibility (RSD between 4 and 11%) in both SIM and MRM modes, and detection limits down to ng/g. The combination of MS/MS and in-source CID allowed confirmation of the presence of CPs in environmental samples.     

Characterization of fatty acid esters of okadaic acid and related toxins in blue mussels (Mytilus edulis) from Norway

Marine algal toxins of the okadaic acid group can occur as fatty acid esters in blue mussels, and are commonly determined indirectly by transformation to their parent toxins by alkaline hydrolysis. Some data are available regarding the identity of the fatty acid esters, mainly of palmitic acid (16:0) derivatives of okadaic acid (OA), dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2). Other fatty acid derivatives have been described, but with limited mass spectral data. In this paper, the mass spectral characterization of the [M-H](-) and [M+Na](+) ions of 16 fatty acid derivatives of each of OA, DTX1 and DTX2 is presented. The characteristic fragmentation of [M+Na](+) ions of OA analogues provided a useful tool for identifying these, and has not been described previously. In addition, a set of negative ion multiple reaction monitoring (MRM) methods was developed for direct determination of 16 fatty acid esters of OA, 16 fatty acid esters of DTX1 and 16 fatty acid esters of DTX2 in shellfish extracts. The MRM methods were employed to study the profiles of fatty acid esters of OA analogues in blue mussels and to compare these with fatty acid ester profiles reported for other groups of marine algal toxins.     

Structural characterization of phosphatidyl-<Emphasis Type="Italic">myo</Emphasis>-inositol mannosides from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> bacillus calmette guérin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. I. PIMs and lyso-PIMs

We described a multiple-stage ion-trap mass spectrometric approach to characterize the structures of phosphatidylinositol and phosphatidyl-myoinositol mannosides (PIMs) in a complex mixture isolated from Mycobacterium bovis Bacillus Calmette Guérin. The positions of the fatty acyl substituents of PIMs at the glycerol backbone can be easily assigned, based on the findings that the ions arising from losses of the fatty acid substituent at sn-2 as molecules of acid and of ketene, respectively (that is, the [M - H - R(2)CO(2)H](-) and [M - H - R(2)CHCO](-) ions), are respectively more abundant than the ions arising from the analogous losses at sn-1 (that is, the [M - H - R(1)CO(2)H](-) and [M - H - R(1)CHCO](-) ions) in the MS(2) product-ion spectra of the [M - H](-) ions desorbed by electrospray ionization (ESI). Further dissociation of the [M - H - R(2)CO(2)H](-) and [M - H - R(1)CO(2)H](-) ions gives rise to a pair of unique ions corresponding to losses of 74 and 56 Da (that is, [M - H - R(x)CO(2)H - 56](-) and [M - H - R(x)CO(2)H - 74](-) ions, x = 1, 2), respectively, probably arising from various losses of the glycerol. The profile of the ion-pair in the MS(3) spectrum of the [M - H - R(2)CO(2)H](-) ion is readily distinguishable from that in the MS(3) spectrum of the [M - H - R(1)CO(2)H](-) ion and thus the assignment of the fatty acid substituents at the glycerol backbone can be confirmed. The product-ion spectra of the [M - H](-) ions from 2-lyso-PIM and from 1-lyso-PIM are discernible and both spectra contain a unique ion that arises from primary loss of the fatty acid substituent at the glycerol backbone, followed by loss of a bicyclic glycerophosphate ester moiety of 136 Da. The combined structural information from the MS(2) and MS(3) product-ion spectra permit the complex structures of PIMs that consist of various isomers to be unveiled in detail.     

Synthesis of derivatives of 4-alkylthiouracil,cytosine, pyrido[2,3-d]pyrimidine,and pyrimido[4,5-d]pyrimidine from the N,S- and N,N-acetals of diacetylketene and isocyanates

The action of alkyl and aryl isocyanates on the N,S-acetals of diacetylketene leads to the formation of 4-alkylthio-5-acetyl-1-alkyl(aryl)-6-methyl-1H-pyrimidin-2-ones (derivatives of 4-alkylthiouracils). The reaction of the synthesized thiouracils with amines or the reaction of the N,N-acetals of diacetylketene (N,N-ADK) with an equi-molar amount of aryl isocyanates leads to the formation of substituted 4-amino-5-acetyl-1H-pyrimidin-2-ones (derivatives of cytosine). From the latter and isocyanates or directly from N,N-ADK and an excess of the isocyanate, derivatives of 4-methylene-1H,3H, 4H-pyrimido[4,5-d]pyrimidine-2,7-dione were obtained. The exception was the condensation of 3-[N-(4,6-dimethyl-2-pyrimidinyl)diaminomethylene]pentane-2,4-dione with aryl isocyanates, which led to 3H,8H-pyrido[2,3-d]pyrimidine-2,5-diones.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 11, pp. 2593–2599, November, 1991.     

Quantification of [Dmt1]DALDA in ovine plasma by on-line liquid chromatography/quadrupole time-of-flight mass spectrometry

The synthetic peptide [Dmt(1)]DALDA (Dmt-D-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine; 'super-DALDA') is a mu opioid-receptor agonist. On-line liquid chromatography/quadrupole time-of-flight mass spectrometry and the corresponding stable isotope-incorporated synthetic peptide internal standard were used to quantify [Dmt(1)]DALDA that had been extracted from ovine plasma samples. The [M+2H](2+) ion was used to construct the calibration curve, and the product ion was used for verification of the peptide. The detection sensitivity for the [Dmt(1)]DALDA [M+2H](2+) ion was 12.5 fmol and 50 fmol for the m/z 432.3 product ion. The concentration profile of [Dmt(1)]DALDA was determined from a set of ovine plasma samples. The molecular specificity of the peptide quantification was confirmed by tandem mass spectrometry (MS/MS).     

Analysis of N7-(benzo[a]pyrene-6-yl)guanine in urine using two-step solid-phase extraction and isotope dilution with liquid chromatography/tandem mass spectrometry

Analysis of urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a DNA adduct induced by benzo[a]pyrene, may serve as a risk-associated biomarker for exposure to polyaromatic hydrocarbons (PAHs). In this study a highly sensitive and specific analytical method, incorporating on-line sample preparation coupled with isotope-dilution liquid chromatography and tandem mass spectrometry (LC/MS/MS), was developed to quantitate this adduct in human urine. In order to achieve accurate quantitation, 15N5-labeled BP-6-N7Gua was synthesized to serve as the internal standard, and a two-step solid-phase extraction (SPE) procedure using C8 and SCX cartridges was used for sample cleanup. BP-6-7-N7Gua was analyzed using positive ion LC/MS/MS operated in multiple reaction monitoring (MRM) mode. The [M+H]+ ions at m/z 402 and 407 and the common fragment ion of [M+H]+ at m/z 252 were monitored for quantification. The recovery of this analyte after two-step SPE was 90%, and the limit of detection was 2.5 fmol/mL in 10 mL of urine. This highly specific and sensitive method for BP-6-N7Gua in urine may be applied to assess exposure to PAHs in coke-oven workers for future molecular epidemiology studies on health effects of PAHs.     

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.     

Structural characterization of phosphatidyl-<Emphasis Type="Italic">myo</Emphasis>-inositol mannosides from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> bacillus calmette gúerin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. II. Monoacyl- and diacyl-PIMs

The multiple-stage ion-trap mass spectrometric approaches towards to the structural characterization of the monoacyl-PIM (triacylated PIM) and the diacyl-PIM (tetracylated PIM), namely, the PIM (diacylated PIM) consisting of one or two additional fatty acid substituents attached to the glycoside, respectively, were described. While the assignment and confirmation of the fatty acid substituents on the glycerol backbone can be easily achieved by the methods described in the previous article, the identity of the glycoside moiety and its acylation state can be determined by the observation of a prominent acylglycoside ion arising from cleavage of the diacylglycerol moiety ([M - H - diacylglycerol](-)) in the MS(2)-spectra of monoacyl-PIM and diacyl-PIM. The distinction of the fatty acid substituents on the 2-O-mannoside (i.e., R(3)CO(2)H) from that on the inositol (i.e., R(4)CO(2)H) is based on the findings that the MS(3)-spectrum of [M - H - diacylglycerol](-) contains a prominent ion arising from further loss of the fatty acid at the 2-O-mannoside (i.e., the [M - H - diacylglycerol - R(3)CO(2)H](-) ion), while the ion arising from loss of the fatty acid substituent at the inositol (i.e., the [M - H - diacylglycerol - R(4)CO(2)H](-) ion) is of low abundance. The fatty acyl moiety on the inositol can also be identified by the product-ion spectrum from MS(4) of the [M - H - diacylglycerol - R(3)CO(2)H](-) ion, which gives rise to a prominent ion corresponding to loss of R(4)CO(2)H. An [M - H - acylmannose](-) ion was also observed in the MS(2)-spectra and, thus, the identity of the fatty acid substituent attached to 2-O-mannoside can be confirmed. The combined information obtained from the multiple-stage product-ion spectra from MS(2), MS(3), and MS(4) permit the assignment of the complex structures of monoacyl-PIMs and diacyl-PIMs in a mixture isolated from M. bovis Bacillus Calmette Guérin.     

Determination of airborne isocyanates as di-n-butylamine derivatives using liquid chromatography and tandem mass spectrometry

A method for the determination of isocyanates as di-n-butyl amine (DBA) derivatives using tandem mass spectrometry (MS/MS) and electrospray ionisation (ESI) is presented. Multiple-reaction monitoring (MRM) of the protonated molecular ions and corresponding deuterium-labelled d₉-DBA derivatives resulted in selective quantifications with correlation coefficients >0.998 for the DBA derivatives of isocyanic acid (ICA), methyl isocyanate (MIC), ethyl isocyanate (EIC), propyl isocyanate (PIC), phenyl isocyanate (PhI), 1,6-hexamethylene diisocyanate (HDI), 2,4-, 2,6-toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), 4,4′-methylenediphenyl diisocyanate (MDI), 3-ring MDI, 4-ring MDI, HDI-isocyanurate, HDI-diisocyanurate, HDI-biuret and HDI-dibiuret. The instrumental precision for 10 repeated injections of a solution containing 0.1 μg ml⁻¹ of the studied derivatives was <2%. Performing MRM of the product ion [DBA + H]⁺ (m/z = 130) from the protonated molecular ion resulted in the lowest detection limits, down to 10 amol (for TDI). Quantification of concentrations below 10⁻⁶ of the occupational exposure limit (OEL) for TDI during 10 min of air sampling was made possible. In an effort to control the formation of alkali adducts, addition of lithium acetate to the mobile phase and monitoring of lithium adducts was evaluated. Having lithium present in the mobile phase resulted in complete domination of [M + Li]⁺ adducts, but detection limits for the studied compounds were not improved. Different deuterium-labelled derivatives as internal standards were evaluated. (1) DBA derivatives of deuterium-labelled isocyanates (d₄-HDI, d₃-2,4-TDI, d₃-2,6-TDI and d₂-MDI), (2) d₉-DBA derivatives of the corresponding isocyanates and (3) d₁₈-DBA derivatives of the corresponding isocyanates. An increase in number of deuterium in the molecule of the internal standard resulted in an increase in instrumental precision and a decrease in correlation within calibration series.     

Different behavior of dextrans in positive-ion and negative-ion mass spectrometry

A mass spectrometric (MS) comparative study of dextran samples using two different ionization techniques (matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI)) in both positive- and negative-ion modes is reported. The experiments were carried out with two polydisperse dextrans (1000 and 8800 Da) and isomaltotriose. In the positive-ion mode, the expected alkali metal ion adducts of dextrans were observed in both techniques. In contrast, the expected preferential formation of deprotonated molecules [M - H](-) was not confirmed in negative mode MALDI time-of-flight (TOF) MS, where the series of ions [M(x)- H +42](-) or [M(x+1)- H - 120](-), coming either from some addition or fragmentation, were observed. In both ionization techniques, these ions formed the main distributions of dextrans in the negative-ion mode. It seems that the negative molecular ions formed from the alpha1 --> 6 linkage of polyglucose oligomers easily decompose, and the product ions [M - H - 120](-) markedly dominate. The fragmentation experiments and especially the investigation of the fundamental role of the nozzle-skimmer potential in ESI-MS supported our explanation of the observed behavior because its higher values caused higher fragmentation. The experiments with isomaltotriose excluded any addition of 42 Da during the MS procedures, which is not distinguishable from the loss of 120 Da in the case of polydisperse dextrans. MALDI-TOFMS was found to be more sensitive for the detection of higher oligosaccharides and ESI-MS more useful for structural studies.     

Structural characterization of glycoporphyrins by electrospray tandem mass spectrometry

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.     

Tautomerization of methyldiazene to formaldehyde-hydrazone in ruthenium and osmium complexes

Mixed-ligand hydrazine complexes [M(CO)(RNHNH2)P4](BPh4)2 (1, 2) [M = Ru, Os; R = H, CH3, C6H5; P = P(OEt)3] with carbonyl and triethyl phosphite were prepared by allowing hydride [MH(CO)P4]BPh4 species to react first with HBF4.Et2O and then with hydrazines. Depending on the nature of the hydrazine ligand, the oxidation of [M(CO)(RNHNH2)P4](BPh4)2 derivatives with Pb(OAc)4 at -30 C gives acetate [M(kappa1-OCOCH3)(CO)P4]BPh4 (3a), phenyldiazene [M(CO)(C6H5N=NH)P4](BPh4)2 (3c, 4c), and methyldiazene [M(CO)(CH3N=NH)P4](BPh4)2 (3b, 4b) derivatives. Methyldiazene complexes 3b and 4b undergo base-catalyzed tautomerization of the CH3N=NH ligand to formaldehyde-hydrazone NH2N=CH2, giving the [M(CO)(NH2N=CH2)P4](BPh4)2 (5, 6) derivatives. Complexes 5 and 6 were characterized spectroscopically and by the X-ray crystal structure determination of the [Ru(CO)(NH2N=CH2)[P(OEt)3]4](BPh4)2 (5) derivative. Acetone-hydrazone [M(CO)[NH2N=C(CH3)2]P4](BPh4)2 (7, 8) complexes were also prepared by allowing hydrazine [M(CO)(NH2NH2)P4](BPh4)2 derivatives to react with acetone.     

Development of a 6-hydroxychroman-based derivatization reagent: application to the analysis of 5-hydroxytryptamine and catecholamines by using high-performance liquid chromatography with electrochemical detection

We developed a novel derivatization reagent, (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA), for electrochemical (EC) detection in HPLC. NPCA was synthesized from (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (alpha-CA), which exhibits intense EC response. NPCA successfully yielded alpha-CA derivatives of primary amines by a two-step derivatization procedure. Following pre-column derivatization with NPCA, a simultaneous determination of alpha-CA derivatives of neuroactive monoamines [dopamine (DA), epinephrine, and 5-hydroxytryptamine (5-HT)], their monoamine oxidase metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid) and their catechol-O-methyltransferase metabolites [3-methoxytyramine (3-MT) and normetanephrine (NMN)] was completely achieved using our HPLC-EC method. Using an HPLC equipped with coulometric electrode-array detection system, the resultant alpha-CA derivatives of NMN, 5-HT, DA and 3-MT showed intense EC responses, that were approximately 1.3, 1.4, 1.1 and 1.4-fold higher than the corresponding native forms, respectively. The detection limits were in the range of approximately 16-60 fmol on column (signal-to-noise ratio 3). The proposed HPLC method was applied to determine 5-HIAA, HVA, alpha-CA-5-HT and alpha-CA-DA in rat urine. As a consequence, these analytes were successfully determined with satisfactory precisions.     

Electrospray ionization mass spectrometry of ginsenosides

Ginsenosides R(b1), R(b2), R(c), R(d), R(e), R(f), R(g1), R(g2) and F(11) were studied systematically by electrospray ionization mass spectrometry in positive- and negative-ion modes with a mobile-phase additive, ammonium acetate. In general, ion sensitivities for the ginsenosides were greater in the negative-ion mode, but more structural information on the ginsenosides was obtained in the positive-ion mode. [M + H](+), [M + NH(4)](+), [M + Na](+) and [M + K](+) ions were observed for all of the ginsenosides studied, with the exception of R(f) and F(11), for which [M + NH(4)](+) ions were not observed. The signal intensities of [M + H](+), [M + NH(4)](+), [M + Na](+) and [M + K](+) ions varied with the cone voltage. The highest signal intensities for [M + H](+) and [M + NH(4)](+) ions were obtained at low cone voltage (15-30 V), whereas those for [M + Na](+) and [M + K](+) ions were obtained at relatively high cone voltage (70-90 V). Collision-induced dissociation yielded characteristic positively charged fragment ions at m/z 407, 425 and 443 for (20S)-protopanaxadiol, m/z 405, 423 and 441 for (20S)-protopanaxatriol and m/z 421, 439, 457 and 475 for (24R)-pseudoginsenoside F(11). Ginsenoside types were identified by these characteristic ions and the charged saccharide groups. Glycosidic bond cleavage and elimination of H(2)O were the two major fragmentation pathways observed in the product ion mass spectra of [M + H](+) and [M + NH(4)](+). In the product ion mass spectra of [M - H](-), the major fragmentation route observed was glycosidic bond cleavage. Adduct ions [M + 2AcO + Na](-), [M + AcO](-), [M - CH(2)O + AcO](-), [M + 2AcO](2-), [M - H + AcO](2-) and [M - 2H](2-) were observed at low cone voltage (15-30 V) only.     

Determination of thiodiglycolic acid in urine by capillary electrophoresis.

A sensitive capillary electrophoretic method was developed for the determination of thiodiglycolic acid (TDA) in urine which avoids the pretreatment of the urine sample. Several carrier electrolytes were examined. The most suitable carrier electrolyte system consisted of potassium hydrogen phthalate (5 mM), 2-(N-morpholino)ethanesulfonic acid (50 mM) and tetradecyltrimethylammonium bromide (0.5 mM), pH 5.2. Ten times diluted fresh midstream void urine was used for the determination. In this way, the concentrations of TDA between 5 and 50 mg/l in undiluted urine samples can be determined.     

Identification of phenolics and nucleoside derivatives in Gastrodia elata by HPLC-UV-MS

An HPLC-UV-MS method for simultaneous identification of predominant phenolics and minor nucleoside derivatives in Gastrodia elata was developed, which was based on their UV and MS characteristics summarized through a series of homemade reference standard experiments. Phenolics showed characteristic UV lambda(max) at 267 nm, [M + NH(4)](+) base peak in positive mode and [M-H](-) base peak in negative mode while nucleosides exhibited UV lambda(max) at 255 nm, [M + H](+), [M-H + 2H(2)O](-) or [M-H + CH(3)COOH](-). Phenolics conjugates mainly underwent the consecutive loss of gastrodin residue (-268 U) and the combined loss of H(2)O and CO(2 )from the citric acid unit under negative MS/MS conditions whereas nucleosides simply lost the ribose (-132 U) under positive MS/MS conditions. According to these characteristics, a special pattern under MS/MS conditions and reported compound data for G. elata in the literature, not only 15 phenolics were identified but also 6 nucleoside derivatives were identified. Among these compounds, seven phenolics and three nucleoside derivatives have not been reported yet from G. elata.     

Forensic confirmatory analysis of ethyl sulfate—A new marker for alcohol consumption—by liquid-chromatography/electrospray ionization/tandem mass spectrometry

Ethyl sulfate (EtS)--a new direct marker for ethanol intake besides ethyl glucuronide (EtG) and others--was detected in urine samples by electrospray ionization tandem mass-spectrometry (LC-ESI-MS/MS). Ethyl sulfate sodium salt was used for method development, yielding a precursor [M - H]- m/z 125 and product ions m/z 97 [HSO4]- and m/z 80 [SO3]-. Pentadeuterated EtS (D5-EtS) was synthesized by esterification of sulfuric acid with anhydrous hexadeutero ethanol ([M - H]- m/z 130, product ions m/z 98 [DSO4]- and m/z 80 [SO3]-). After addition of D5-EtS and D5-EtG, urine samples were analyzed by direct injection into the gradient LC-MS/MS system. Analysis was performed in accordance with forensic guidelines for confirmatory analysis using one precursor and two product ions. EtS has been detected (in addition to EtG) in the urine samples of nine volunteers after drinking sparkling wine containing between 9 and 49 g of ethanol. Both EtS and EtG could be detected up to 36 h after consumption of alcohol. The excretion profile was found to be similar to that of EtG. No EtS was found in teetotalers' urine samples. Method validation parameters are presented. EtS was stable in urine upon storage up to twenty days at room temperature. In addition to EtG, EtS can be used to detect recent alcohol consumption, thus providing a second marker for the time range of up to approximately one day after elimination of ethanol from urine samples. The determination of EtS can be used in addition to EtG as proof of ethanol consumption in workplace monitoring programs.