Pentafluorobenzyl Research Articles

Advances in Derivatization Techniques Enabled by DABCO for Novichok Agent Analysis in Biofluids Using LC-MS.

The characterization of Novichoks (NVs), a new group of nerve agents that have been implicated in two recent poisonings, has not been extensively conducted. Here, we present a novel method for analyzing NV hydrolysates using liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled by pentafluorobenzyl (PFB) derivatization followed by reaction with 1,4-diazabicyclo[2.2.2]octane (DABCO). This approach enabled efficient, simultaneous screening of six NV hydrolysates, with 1-2 orders improvement in the limit of detection in relation to that achieved through previous methods. A straightforward pretreatment using DABCO and filtration was employed for biological samples, mitigating instrument damage and allowing LC-MS/MS after a reaction with highly hydrophobic PFB bromide (PFBBr). In addition, the use of pralidoxime (PAM) significantly enhanced the detection of NV hydrolysates from NV-surrogate-spiked serum. While PAM is not a proven NV antidote, its effectiveness as an analytical reagent to aid in the detection of NV hydrolysates was demonstrated for the first time. Understanding the proposed mechanism of DABCO-mediated derivatization reagent removal in this research could broaden the range of compounds amenable to derivatization LC, thereby enhancing the capabilities of conventional derivatization techniques.

GC-MS Studies on the Conversion and Derivatization of γ-Glutamyl Peptides to Pyroglutamate (5-Oxo-Proline) Methyl Ester Pentafluoropropione Amide Derivatives.

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, γ-Glu-Cys-Gly) is the most abundant intra-cellular dicarboxylic tripeptide with multiple physiological roles. In biological samples, glutathione exists in its reduced form GSH and in two stable oxidized forms, i.e., in its symmetric disulfide form GSSG and as S-glutathionyl residue in proteins. S-Glutathionylation is a post-translational modification, which is involved in several pathophysiological processes, including oxidative stress. The GSH-to-GSSG molar ratio is widely used as a measure of oxidative stress. γ-Glutamyl is the most characteristic structural moiety of GSH. We performed gas chromatography-mass spectrometry (GC-MS) studies for the development of a highly specific qualitative and quantitative method for γ-glutamyl peptides. We discovered intra-molecular conversion of GSH, GSSG, γ-Glu-Cys and of ophthalmic acid (OPH; γ-glutamyl-α-amino-n-butyryl-glycine) to pyroglutamate (pGlu; 5-oxo-proline, also known as pidolic acid) during their derivatization with 2 M HCl/CH3OH (60 min, 80 °C). For GC-MS analysis, the methyl esters (Me) were further derivatized with pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v; 30 min, 65 °C) to their PFP derivatives. At longer reaction times, pGlu is hydrolyzed to Glu. Internal standards were prepared by derivatizing GSH, GSSG, γ-Glu-Cys and OPH in 2 M HCl/CD3OD. Quantification of the Me-PFP derivative of pGlu was performed in the electron-capture negative-ion chemical ionization (ECNICI) mode by selected-ion monitoring (SIM) of the mass-to-charge (m/z) ions 269 for unlabeled pGlu (d0Me-PFP-pGlu) and m/z 272 for the in situ prepared deuterium-labeled pGlu (d3Me-PFP-pGlu). Although not inherent to the analysis of small peptides, the present GC-MS method is useful to study several biochemical aspects of GSH. Using pentafluorobenzyl bromide (PFB-Br) as the derivatization reagent, we found that synthetic pGlu is converted in aqueous acetone (60 min, 50 °C) into its pentafluorobenzyl (PFB) ester (PFB-pGlu). This derivatization procedure is useful for the GC-MS analysis of free pGlu in the ECNICI mode. Quantitative analysis of PFB-pGlu by GC-MS requires the use of stable-isotope labeled analogs of pGlu as an internal standard.

Pentafluorobenzylation and detection of sodium monofluoroacetate (compound 1080) in veterinary samples using gas chromatography/tandem quadrupole mass spectrometry with multiple reaction monitoring.

The analytical detection of chemical residues from sodium monofluoroacetate (MFA) ingestion in targeted predatory wildlife and in pesticide misuse incidents perpetrated against nuisance companion animals remains a concern in veterinary forensic toxicology. There is a current need for chemically stable sample extracts with reliable and specific diagnostic methods for trace quantities in diverse diagnostic matrices. Biphasic pentafluorobenzylation provided a simple combined extraction and derivatization procedure for removing MFA in a chemically stable form from a complex matrix such as stomach contents. Analysis of the derivatized extract using gas chromatography/tandem quadrupole mass spectrometry (GC/MS/MS) with multiple reaction monitoring (MRM) approaches specific to MFA provided greater specificity than simple scan or selected ion monitoring approaches. Collision-induced dissociation in GC/MS/MS showed that pentafluorobenzyl (PFB)-derivatized MFA (M+ m/z 258) generated m/z 258➔130, 149, 161, 177, 178, 180.1, and 181.1 transitions. Of these, the transition m/z 258➔181 provided a peak for quantitation, whereas m/z 258➔161 and 258➔178 provided specificity for qualifying MFA. Similarly, PFB-derivatized 2-chloropropionic acid (M+ m/z 288) was used as an internal standard, which generated m/z 288➔181 and 161. Of these, the transition m/z 288➔181 provided a peak for quantitation, whereas m/z 288➔161 and 181➔161 served to qualify the internal standard. The method was validated with a calculated limit of detection of 0.35 ppm and limit of quantitation of 1.09 ppm MFA. The method should have adequate sensitivity and reliability for veterinary toxicology labs analyzing specimens from animals poisoned by this predacide.

One-pot synthesis of amphiphilic multiblock poly(2-oxazoline)s via para-fluoro-thiol click reactions

A clickable initiator, pentafluoro benzyl bromide, has been investigated for the cationic ring opening polymerization of poly(2-oxazolines).

GC-NICI-MS analysis of acetazolamide and other sulfonamide (R-SO2-NH2) drugs as pentafluorobenzyl derivatives [R-SO2-N(PFB)2] and quantification of pharmacological acetazolamide in human urine

Acetazolamide (molecular mass (MM), 222) belongs to the class of sulfonamides (R-SO2-NH2) and is one of the strongest pharmacological inhibitors of carbonic anhydrase activity. Acetazolamide is excreted unchanged in the urine. Here, we report on the development, validation and biomedical application of a stable-isotope dilution GC-MS method for the reliable quantitative determination of acetazolamide in human urine. The method is based on evaporation to dryness of 50 μL urine aliquots, base-catalyzed derivatization of acetazolamide (d0-AZM) and its internal standard [acetylo-2H3]acetazolamide (d3-AZM) in 30 vol% pentafluorobenzyl (PFB) bromide in acetonitrile (60 min, 30 °C), reconstitution in toluene (200 μL) and injection of 1-μL aliquots. The negative-ion chemical ionization (NICI) mass spectra (methane) of the PFB derivatives contained several intense ions including [M]‒ at m/z 581 for d0-AZM and m/z 584 for d3-AZM, suggesting derivatization of their sulfonamide groups to form N,N-dipentafluorobenzyl derivatives (R-SO2-N(PFB)2), i.e., d0-AZM-(PFB)2 and d3-AZM-(PFB)2, respectively. Quantification was performed by selected-ion monitoring of m/z 581 and 83 for d0-AZM-(PFB)2 and m/z 584 and 86 for d3-AZM-(PFB)2. The limits of detection and quantitation of the method were determined to be 300 fmol (67 pg) and 1 μM of acetazolamide, respectively. Intra- and inter-assay precision and accuracy for acetazolamide in human urine samples in pharmacologically relevant concentration ranges were determined to be 0.3%–4.2% and 95.3%–109%, respectively. The method was applied to measure urinary acetazolamide excretion after ingestion of a 250 mg acetazolamide-containing tablet (Acemit®) by a healthy volunteer. Among other tested sulfonamide drugs, methazolamide (MM, 236) was also found to form a N,N-dipentafluorobenzyl derivative, whereas dorzolamide (MM, 324) was hardly detectable. No GC-MS peaks were obtained from the PFB bromide derivatization of hydrochlorothiazide (MM, 298), xipamide (MM, 355), indapamide and metholazone (MM, 366 each) or brinzolamide (MM, 384). We demonstrate for the first time that sulfonamide drugs can be derivatized with PFB bromide and quantitated by GC-MS. Sulfonamides with MM larger than 236 are likely to be derivatized by PFB bromide but to lack thermal stability.

Investigation of NG-hydroxy-l-arginine interference in the quantitative determination of nitrite and nitrate in human plasma and urine by GC-NICI-MS

NG-Hydroxy-l-arginine (NOHA) is the intermediate product of the conversion of l-arginine to l-citrulline and nitric oxide (NO) by nitric oxide synthase (NOS). NO is further oxidized to nitrite and nitrate which circulate in the blood and are excreted in the urine. Nitrite and nitrate may therefore serve as surrogates of NO synthesis. NOHA has been reported to occur in various cells and in blood of animals and humans. The concentration of nitrite in the circulation is comparable to the concentration of NOHA in plasma and serum of humans and laboratory animals. NOHA is a relatively unstable compound and the interaction of its NG-hydroxy group with redox active species or during sample treatment such as derivatization in the heat may yield N-containing compounds including nitrite and nitrate. In theory, NOHA may interfere with the analysis of nitrite and nitrate. In the present study, we investigated a possible interference of synthetic NOHA (0–400 μM) with the gas chromatography-negative ion chemical ionization–mass spectrometry (GC-NICI-MS) method of analysis of circulating and urinary nitrite and nitrate involving derivatization with pentafluorobenzyl (PFB) bromide in aqueous acetone at 50 °C for 5 min (nitrite) or for 60 min (nitrite and nitrate). Our results show that NOHA does not interfere with the measurement of nitrite and nitrate in human plasma and urine by this method at concentrations up to 400 μM.

Glucosamine-Based Supramolecular Nanotubes for Human Mesenchymal Cell Therapy.

Herein, we demonstrate an example of glucosamine-based supramolecular hydrogels that can be used for human mesenchymal cell therapy. We designed and synthesized a series of amino acid derivatives based on a strategy of capping d-glucosamine moiety at the C-terminus and fluorinated benzyl group at the N-terminus. From a systematic study on chemical structures, we discovered that the glucosamine-based supramolecular hydrogel [pentafluorobenzyl (PFB)-F-Glu] self-assembled with one-dimensional nanotubular structures at physiological pH. The self-assembly of a newly discovered PFB-F-Glu motif is attributed to the synergistic effect of π-π stacking and extensive intermolecular hydrogen bonding network in aqueous medium. Notably, PFB-F-Glu nanotubes are proven to be nontoxic to human mesenchymal stem cells (hMSCs) and have been shown to enhance hMSC proliferation while maintaining their pluripotency. Retaining of pluripotency capabilities provides potentially unlimited source of undifferentiated cells for the treatment of future cell therapies. Furthermore, hMSCs cultured on PFB-F-Glu are able to secrete paracrine factors that downregulate profibrotic gene expression in lipopolysaccharide-treated human skin fibroblasts, which demonstrates that PFB-F-Glu nanotubes have the potential to be used for wound healing applications. Overall, this article addresses the importance of chemical design to generate supramolecular biomaterials for stem cell therapy.

Comparison of derivatization/ionization techniques for liquid chromatography tandem mass spectrometry analysis of oxylipins

The performance of two derivatization and ionization techniques for the quantitative reversed phase liquid chromatography (LC)- mass spectrometry (MS) analysis of hydroxy fatty acids (OH-PUFA) in plasma was evaluated: One used AMPP (N-(4-aminomethylphenyl)pyridinium chloride) leading to a positive charged amid-derivate which can be detected by electrospray ionization (ESI)-MS. Second yielded penta fluorobenzyl bromide (PFB) ester derivates allowing detection in electron capture atmospheric pressure chemical ionization (ecAPCI)-MS. The sensitivity of detection of a comprehensive set of hydroxy fatty acids of n6- and n3- poly unsaturated fatty acids was investigated. On the SCIEX3200 MS the applied PFB derivatization led to poor limits of detection (LOD) of 10–100nM (0.1–1pmol/0.03–0.3ng on column). By contrast, AMPP derivatization led to a similar sensitivity compared to the standard ESI(-) of non derivatized analytes (LOD about 1nM (10fmol/3pg on column)). For several analytes, including 9-HETE, 11-HETE and 17-HDHA the AMPP derivatization improved sensitivity enabling their detection in human plasma. However, precision was reduced by AMPP derivatization and variation in IS recovery indicated a strong matrix influence on the MS-signal. In sum, with the instrumentation used, neither of these derivatization methods improves in our hands the LC–MS based quantification of oxylipins.

GC-ECNICI-MS analysis of S-nitrosothiols and nitroprusside after treatment with aqueous sulphide (S2-) and derivatization with pentafluorobenzyl bromide: Evidence of S-transnitrosylation and formation of nitrite and nitrate.

A GC-MS method is reported for the quantitative analysis of S-nitrosothiols (RSNO) derived from endogenous low- and high-molecular mass thiols (RSH) including hemoglobin, cysteine, glutathione, N-acetylcysteine, and the exogenous N-acetylcysteine ethyl ester. The method is based on the conversion of RSNO to nitrite by aqueous Na2S (S2-). 15N-Labelled analogs (RS15NO) or 15N-labelled nitrite and nitrate were used as internal standards. The nitrite (14NO2- and 15NO2-) and nitrate (O14NO2- and O15NO2- anions were derivatised by pentafluorobenzyl (PFB) bromide (PFB-Br) in aqueous acetone and their PFB derivatives were separated by gas chromatography. After electron-capture negative-ion chemical ionization, the anions were separated by mass spectrometry and detected by selected-ion monitoring of m/z 46 for 14NO2-, m/z 47 for 15NO2-, m/z 62 for O14NO2-, and m/z 63 for O15NO2-. The expected thionitrites (-S14NO and -S15NO) were not detected, suggesting that they are intermediates and rapidly exchange their S by O from water, presumably prior to PFB-Br derivatization. The reaction of S2- with RSNO and sodium nitroprusside (SNP) resulted in the formation of nitrite and nitrate as the major and minor reaction products, respectively. The novel Na2S procedure was compared with established procedures based on the use of aqueous HgCl2 or cysteine/Cu2+ reagents to convert the S-nitroso group to nitrite. Our results provide evidence for an equilibrium S-transnitrosylation reaction between S2- with RSNO in buffered solutions of neutral pH. Use of Na2S in molar excess over RSNO shifts this reaction to the right, thus allowing almost complete conversion of RSNO to nitrite and nitrate. The Na2S procedure should be useful for the quantitative determination of RSNO as nitrite and nitrate after PFB-Br derivatization and GC-MS analysis. The Na2S procedure may also contribute to explore the complex reactions of S2- with RSNO, SNP and other NO-containing compounds.

Cross-validated stable-isotope dilution GC-MS and LC-MS/MS assays for monoacylglycerol lipase (MAGL) activity by measuring arachidonic acid released from the endocannabinoid 2-arachidonoyl glycerol.

2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC-MS and LC-MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d8-2AG; 10μM) as the MAGL substrate and measure deuterium-labeled AA (d8-AA; range 0-1μM) as the MAGL product. Unlabelled AA (d0-AA, 1μM) serves as the internal standard. d8-AA and d0-AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC-MS/MS analysis or in anhydrous acetonitrile for GC-MS analysis. LC-MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M-H]-→[M-H - CO2]-, i.e., m/z 311→m/z 267 for d8-AA and m/z 303→m/z 259 for d0-AA. Prior to GC-MS analysis d8-AA and d0-AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC-MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M-PFB]-, i.e., m/z 311 for d8-AA and m/z 303 for d0-AA. The GC-MS and LC-MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0-1μM) of d8-AA measured by LC-MS/MS (y) and that by GC-MS (x) revealed a straight line (r2=0.9848) with the regression equation y=0.003+0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination of d8-AA produced from d8-2AG by hepatic MAGL activity. The formation of d8-prostaglandin E2 by the consecutive catalytic action of recombinant MAGL on d8-2AG and recombinant cyclooxygenase-2 (COX) on d8-AA was demonstrated by GC-MS/MS.

Simultaneous pentafluorobenzyl derivatization and GC-ECNICI-MS measurement of nitrite and malondialdehyde in human urine: Close positive correlation between these disparate oxidative stress biomarkers.

Urinary nitrite and malondialdehyde (MDA) are biomarkers of nitrosative and oxidative stress, respectively. At physiological pH values of urine and plasma, nitrite and MDA exist almost entirely in their dissociated forms, i.e., as ONO- (ONOH, pKa=3.4) and -CH(CHO)2 (CH2(CHO)2, pKa=4.5). Previously, we reported that nitrite and MDA react with pentafluorobenzyl (PFB) bromide (PFB-Br) in aqueous acetone. Here, we report on the simultaneous derivatization of nitrite and MDA and their stable-isotope labeled analogs O15NO- (4μM) and CH2(CDO)2 (1μM or 10μM) with PFB-Br (10μL) to PFBNO2, PFB15NO2, C(PFB)2(CHO)2), C(PFB)2(CDO)2 by heating acetonic urine (urine-acetone, 100:400μL) for 60min at 50°C. After acetone evaporation under a stream of nitrogen, derivatives were extracted with ethyl acetate (1mL). A 1-μL aliquot of the ethyl acetate phase dried over anhydrous Na2SO4 was injected in the splitless mode for simultaneous GC-MS analysis in the electron capture negative-ion chemical ionization mode. Quantification was performed by selected-ion monitoring (SIM) the anions [M-PFB]-m/z 46 for ONO-, m/z 47 for O15NO-, m/z 251 for -C(PFB)(CHO)2, and m/z 253 for -C(PFB)(CDO)2. The retention times were 3.18min for PFB-ONO2/PFB-O15NO2, and 7.13min for -C(PFB)(CHO)2/-C(PFB)(CDO)2. Use of CH2(CDO)2 at 1μM but not at 10μM was associated with an unknown interference with the C(PFB)2(CDO)2 peak. Endogenous MDA can be quantified using O15NO- (4μM) and CH2(CDO)2 (10μM) as the internal standards. The method is also useful for the measurement of nitrate and creatinine in addition to nitrite and MDA. Nitrite and MDA were measured by this method in urine of elderly healthy subjects (10 females, 9 males; age, 60-70 years; BMI, 25-30kg/m2). Creatinine-corrected excretion rates did not differ between males and females for MDA (62.6 [24-137] vs 80.2 [52-118]nmol/mmol, P=0.448) and for nitrite (102 [71-174] vs. 278 [110-721]nmol/mmol P=0.053). We report for the first time a close correlation (r=0.819, P<0.0001) between MDA and nitrite in human urine. This correlation is assumed to be due to involvement of myeloperoxidase which catalyzes the formation of hypochlorite (-OCl) from chloride and hydrogen peroxide. In turn, hypochlorite reacts both with nitrite and with polyunsaturated fatty acids such as arachidonic acid, with the later reaction generating MDA. The proposed mechanisms are supported by the literature but remain to be fully explored.

GC-ECNICI-MS/MS of eicosanoids as pentafluorobenzyl-trimethylsilyl (TMS) derivatives: Evidence of CAD-induced intramolecular TMS ether-to-ester rearrangement using carboxy-18O-labelled eicosanoids and possible implications in quantitative analysis.

GC-MS and GC-MS/MS of pentafluorobenzyl (PFB) ester trimethylsilyl (TMS) ether (PFB-TMS) derivatives of hydroxylated long-chain fatty acids including arachidonic acid metabolites, the eicosanoids, in the electron-capture negative-ion chemical ionization (ECNICI) mode are the most sensitive and accurate approaches to quantify carboxyl groups-containing compounds in complex biological fluids such as plasma and urine. Under ECNICI conditions, PFB-TMS derivatives of eicosanoids ionize to form very few ions, with the carboxylates [M-PFB]- being typically the most intense. Less intense ions may be additionally formed by consecutive neutral loss (NL) of trimethylsilanol (TMSOH, 90Da) groups ([M-PFB-(TMSOH)n]-). By using [1,1-18O2]- and [1,ω-18O2]-eicosanoids, we studied ion processes following collisionally activated dissociation (CAD) of the precursor ions [M-PFB]-. We found that CAD resulted in formation of product ions due to NL of a TMS18OH (92Da) group in monocarboxylic and of a PFB18OH (200Da) group in dicarboxylic eicosanoids. TMS18OH NL implies an intra-molecular transfer of the TMS group from hydroxyl groups to their carboxylate anions [M-PFB]-. From a mechanistic point of view, this rearrangement may explain formation of unique product ions in GC-MS/MS of eicosanoids under ECNICI conditions. From the quantitative point of view, quantification by GC-MS/MS of product ions due to [M-PFB-(TMSOH)n]- and [M-PFB-TMS18OH-(TMSOH)n-1]-would reveal incorrect data, if [1,1-18O2]-eicosanoids are used as internal standards and if no correction for the 18O-loss is performed. In 18O-labelled dicarboxylic eicosanoids, such as the major urinary metabolite (MUM) of E prostaglandins, i.e., [1,ω-18O2]-PGE-MUM), no TMS ester/TMS ether rearrangement was observed. Yet, 18O-loss occurred upon CAD of [M-PFB]- due to NL of PFB18OH (200Da). In both cases the extent of 18O-loss needs to be determined and considered for accurate quantification of monocarboxylic acids such as 8-isoprostaglandin F2α (8-iso-PGF2α) and dicarboxylic eicosanoids such as PGE-MUM.

GC-MS and GC-MS/MS measurement of ibuprofen in 10-μL aliquots of human plasma and mice serum using [α-methylo-2H3]ibuprofen after ethyl acetate extraction and pentafluorobenzyl bromide derivatization: Discovery of a collision energy-dependent H/D isotope effect and pharmacokinetic application to inhaled ibuprofen-arginine in mice.

GC-MS and GC-MS/MS methods were developed and validated for the quantitative determination of ibuprofen (d0-ibuprofen), a non-steroidal anti-inflammatory drug (NSAID), in human plasma using α-methyl-2H3-4-(isobutyl)phenylacetic acid (d3-ibuprofen) as internal standard. Plasma (10μL) was diluted with acetate buffer (80μL, 1M, pH 4.9) and d0- and d3-ibuprofen were extracted with ethyl acetate (2×500μL). After solvent evaporation d0- and d3-ibuprofen were derivatized in anhydrous acetonitrile by using pentafluorobenzyl (PFB) bromide and N,N-diisopropylethylamine as the base catalyst. Under electron-capture negative-ion chemical ionization (ECNICI), the PFB esters of d0- and d3-ibuprofen readily ionize to form their carboxylate anions [M-PFB]- at m/z 205 and m/z 208, respectively. Collision-induced dissociation (CID) of m/z 205 and m/z 208 resulted in the formation of the anions at m/z 161 and m/z 164, respectively, due to neutral loss of CO2 (44 Da). A collision energy-dependent H/D isotope effect was observed, which involves abstraction/elimination of H- from d0-ibuprofen and D- from d3-ibuprofen and is minimum at a CE value of 5eV. Quantitative GC-MS determination was performed by selected-ion monitoring of m/z 205 and m/z 208. Quantitative GC-MS/MS determination was performed by selected-reaction monitoring of the mass transitions m/z 205 to m/z 161 for d0-ibuprofen and m/z 208 to m/z 164 for d3-ibuprofen. In a therapeutically relevant concentration range (0-1000μM) d0-ibuprofen added to human plasma was determined with accuracy (recovery, %) and imprecision (relative standard deviation, %) ranging between 93.7 and 110%, and between 0.8 and 4.9%, respectively. GC-MS (y) and GC-MS/MS (x) yielded almost identical results (y=4.00+0.988x, r2=0.9991). In incubation mixtures of arachidonic acid (10μM), d3-ibuprofen (10μM) or d0-ibuprofen (10μM) with ovine cyclooxygenase (COX) isoforms 1 and 2, the concentration of d3-ibuprofen and d0-ibuprofen did not change upon incubation at 37°C up to 60min. The trough pharmacokinetics of an inhaled arginine-containing ibuprofen preparation in mice was studied after once-daily treatment (0.0, 0.07, 0.4 and 2.5mg/kg body weight) for three days. A linear relationship between ibuprofen concentration in serum (10μL) and administered dose 24h after the last drug administration was observed.

Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings.

Creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous substances. Nitrite (ONO-) and nitrate (ONO2-) are metabolites of nitric oxide (NO), a signalling molecule with multiple biological functions. Under certain and standardized conditions, the concentration of nitrate in the urine is a suitable measure of whole body NO synthesis. The urinary nitrate-to-nitrite molar ratio (UNOxR) may indicate nitrite-dependent renal carbonic anhydrase (CA) activity. In clinical studies, urine is commonly collected by spontaneous micturition. In those cases the nitrate and nitrite excretion must be corrected for creatinine excretion. Pentafluorobenzyl (PFB) bromide (PFB-Br) is a useful derivatization reagent of numerous inorganic and organic compounds, including urinary nitrite, nitrate and creatinine, for highly sensitive and specific quantitation by GC-MS. Here, we report on the simultaneous PFB-Br derivatization (60min, 50°C) of ONO-, O15NO-, ONO2-, O15NO2-, creatinine (do-Crea) and [methylo-2H3]creatinine (d3-Crea) in acetonic dilutions of native human urine and plasma samples (4:1, v/v) and their simultaneous quantification by GC-MS as PFBNO2, PFB15NO2, PFBONO2, PFBO15NO2, do-Crea-PFB and d3-Crea-PFB, respectively. Electron capture negative-ion chemical ionization (ECNICI) of these derivatives generates anions due to [M-PFB]-, i.e., the starting analytes. Quantification is performed by selected-ion monitoring (SIM) of m/z 46 (ONO-), m/z 47 (O15NO-), m/z 62 (ONO2-), m/z 63 (O15NO2-), m/z 112 (do-Crea), and m/z 115 (d3-Crea). Retention times were 2.97min for PFB-ONO2/PFB-O15NO2, 3.1min for PFB-NO2/PFB-15NO2, and 6.7min for do-Crea-PFB/d3-Crea-PFB. We used this method to investigate the effects of long-term oral NaNO3 or NaCl (serving as placebo) supplementation (each 0.1mmol/kg body weight per day for 3 weeks) on creatinine excretion and UNOxR in 17 healthy young men. Compared to NaCl (n=8), NaNO3 (n=9) supplementation increased UNOxR (1709±355 vs. 369±77, P<0.05). Creatinine excretion did not differ between the groups (6.67±1.34mM vs. 5.72±1.27mM, P=0.57). The method is also applicable to human plasma. In 78 adults patients newly diagnosed for cerebrovascular disease (CVD), there was a close correlation (r=0.9833) between the creatinine concentrations measured in plasma by GC-ECNICI-MS and those measured in serum by an enzymatic assay. Creatinine-corrected plasma nitrate and nitrite concentrations (P=0.035 and P=0.004, respectively) but not their concentrations (P=0.68 and P=0.40, respectively) differ between male (n=54) and female (n=24) CVD patients. No such differences were found between preterm newborn boys (n=25) and girls (n=22). Like in urine, circulating creatinine may be useful to correct for gender-specific differences in plasma nitrite and nitrate in adults. Chronic NaNO3 supplementation to healthy young men does not affect renal CA-dependent nitrite excretion or creatinine synthesis and excretion.

Quantification of Polyfunctional Thiols in Wine by HS-SPME-GC-MS Following Extractive Alkylation.

Analyses of key odorous polyfunctional volatile thiols in wines (3-mercaptohexanol (3-MH), 3-mercaptohexylacetate (3-MHA), and 4-mercapto-4-methyl-2-pentanone (4-MMP)) are challenging due to their high reactivity and ultra-trace concentrations, especially when using conventional gas-chromatography electron impact mass spectrometry (GC-EI-MS). We describe a method in which thiols are converted to pentafluorobenzyl (PFB) derivatives by extractive alkylation and the organic layer is evaporated prior to headspace solid phase microextraction (HS-SPME) and GC-EI-MS analysis. Optimal parameters were determined by response surface area modeling. The addition of NaCl solution to the dried SPME vials prior to extraction resulted in up to less than fivefold improvement in detection limits. Using 40 mL wine samples, limits of detection for 4-MMP, 3-MH, and 3-MHA were 0.9 ng/L, 1 ng/L, and 17 ng/L, respectively. Good recovery (90%–109%) and precision (5%–11% RSD) were achieved in wine matrices. The new method was used to survey polyfunctional thiol concentrations in 61 commercial California and New York State wines produced from V. vinifera (Riesling, Gewürztraminer, Cabernet Sauvignon, Sauvignon blanc and non-varietal rosé wines), V. labruscana (Niagara), and Vitis spp. (Cayuga White). Mean 4-MMP concentrations in New York Niagara (17 ng/L) were not significantly different from concentrations in Sauvignon blanc, but were significantly higher than 4-MMP in other varietal wines.

GC–MS/MS and LC–MS/MS studies on unlabelled and deuterium-labelled oleic acid (C18:1) reactions with peroxynitrite (ONOO−) in buffer and hemolysate support the pM/nM-range of nitro-oleic acids in human plasma

Oleic acid (cis-9,10-octadecenoic acid) is the most abundant monounsaturated fatty acid in human blood. Peroxynitrite (ONOO(-)) is a short-lived species formed from the reaction of nitric oxide (NO) and superoxide (O2(-)). Peroxynitrite is a potent oxidizing and moderate nitrating agent. We investigated reactions of unlabelled and deuterium labelled oleic acid in phosphate buffered saline (PBS) and lysed human erythrocytes with commercially available sodium peroxynitrite (Na(+)ONOO(-)). Non-derivatized reaction products were analyzed by spectrophotometry, HPLC with UV absorbance detection, and LC-MS/MS electrospray ionization in the negative-ion mode. Reaction products were also analyzed by GC-MS/MS in the electron capture negative-ion chemical ionization mode after derivatization first with pentafluorobenzyl (PFB) bromide and then with N,O-bis(trimethylsilyl)trifluoroacetamide. Identified oleic acid reaction products in PBS and hemolysate include cis-9,10-epoxystearic acid and trans-9,10-epoxystearic acid (about 0.1% with respect to oleic acid), threo- and erythro-9,10-dihydroxy-stearic acids. Vinyl nitro-oleic acids, 9-nitro-oleic acid (9-NO2OA) and 10-nitro-oleic acid (10-NO2OA), or other nitro-oleic acids were not found to be formed from the reaction of oleic acid with peroxynitrite in PBS or hemolysate. Our in vitro study suggests that peroxynitrite oxidizes but does not nitrate oleic acid in biological samples. Unlike thiols and tyrosine, oleic acid is not susceptible to peroxynitrite. GC-MS/MS analysis of PFB esters is by far more efficient than LC-MS/MS analysis of non-derivatized oleic acid and its derivates. Our in vitro results support our previous in vivo findings that nitro-oleic acid plasma concentrations of healthy and diseased subjects are in the pM/nM-range.

An improved mass spectrometry-based measurement of NO metabolites in biological fluids

Assessment of NO metabolism in vivo relies on the accurate measurement of its metabolites nitrite (NO2−), nitrate (NO3−), and nitrosothiols (RSNOs) in biological fluids. We report a sensitive method to simultaneously determine NO2− and NO3− in biological matrixes. Tetraoctylammonium was used to catalyze the complete conversion of NO2− and NO3− to stable pentafluorobenzyl (PFB) derivatives directly from aqueous acetone medium before gas chromatography and negative-ion chemical ionization mass spectrometry (GC/NICI/MS). This catalyst dramatically improved the yield of PFB derivatives for NO2− (4.5 times) and NO3− (55 times) compared to noncatalyzed derivatization methods. Analysis was performed using 15N-labeled internal standards by selected-ion monitoring at m/z 46 for fragment NO2− and m/z 47 for its isotope analogue, 15NO2−, and m/z 62 for NO3− and m/z 63 for 15NO3−. This method allowed specific detection of both PFB derivatives over a wide dynamic range with a limit of detection below 4.5 pg for NO2− and 2.5 pg for NO3−. After the specific conversion of RSNOs by HgCl2 to NO2−, this GC/NICI/MS analysis was used to measure RSNOs in plasma. A further comparison with the widely used tri-iodide chemiluminescence (I3−-CL) assay indicated that the GC/MS assay validated the lower physiological RSNO and nitrite levels reported using I3−-CL detection compared with values obtained using UV–photolysis methods. Plasma levels of RSNOs determined by GC/MS and I3−-CL were well correlated (r = 0.8). The improved GC/MS method was successfully used to determine the changes in plasma, urinary, and salivary NO2− and NO3− as well as plasma RSNOs in humans after either a low-NO3− or a high-NO3− meal.

Mammalian fatty acid synthase activity from crude tissue lysates tracing 13C-labeled substrates using gas chromatography–mass spectrometry

Fatty acid synthase (FASN or FAS, EC 2.3.1.85) is the sole mammalian enzyme to synthesize fatty acids de novo from acetyl- and malonyl-coenzyme A (CoA) esters. This article describes a new method that directly quantifies uniformly labeled 13C16-labeled palmitate ([13C16]palmitate) by tracing [13C2]acetyl-CoA and [13C3]malonyl-CoA using an in vitro FASN assay. This method used gas chromatography–mass spectrometry (GC–MS) to detect [13C16]palmitate carboxylate anions (m/z 271) of pentafluorobenzyl (PFB) derivatives and was highly sensitive at femtomole quantities. Uniformly incorporated [13C16]palmitate was the primary product of both recombinant and crude tissue lysate FASN. Quantification of FASN protein within crude tissue lysates ensured equal FASN amounts, preserved steady-state kinetics, and enabled calculation of FASN-specific activity. FASN activity determined by [13C16]palmitate synthesis was consistent with values obtained from β-nicotinamide adenine dinucleotide 2′-phosphate (NADPH) oxidation assays. Analysis of FASN activity from tissue extracts was not hampered by contaminating enzymes or preexisting fatty acids. Crude mammary gland and liver lysates had significantly different activities at 82 and 65nmolmin−1mg−1, respectively, suggesting that tissue-specific activity levels differ in a manner unrelated to FASN amount. GC–MS quantification of [13C16]palmitate synthesis permits sensitive evaluation of FASN activity from tissues of varied physiological states and of purified FASN activity in the presence of modifying proteins, enzymes, or drugs.

Analysis of Nε‐Ethyllysine in Human Plasma Proteins by Gas Chromatography–Negative Ion Chemical Ionization/Mass Spectrometry as a Biomarker for Exposure to Acetaldehyde and Alcohol

N(ε) -ethyllysine (NEL) is a major stable adduct formed by the reaction of acetaldehyde (AA) with lysine residues in proteins. However, its occurrence and levels in biological specimens and its relationship with AA/alcohol exposure-associated disorders have not been fully elucidated. In this study, we have developed a sensitive and specific method to quantitate NEL levels in human plasma proteins. The method consists of (1) purification of the protein fraction of interest by Sephadex G-15 to remove low molecular substances, (2) hydrolysis of proteins with Pronase E in the presence of stable isotope-labeled internal standards, (3) derivatization of amino acids with pentafluorobenzyl (PFB) bromide, and (4) quantification of the PFB derivatives of NEL and l-lysine using gas chromatography-negative ion chemical ionization/mass spectrometry in a selected ion monitoring mode. Using the above method, the NEL levels in human plasma proteins obtained from 10 each of control subjects and alcoholic patients were measured. NEL was detected in all samples analyzed, the average level of NEL in the plasma proteins of alcoholic patients (1.17±0.36 NEL/1,000 l-lysine) being significantly higher than that of control subjects (0.26±0.07 NEL/1,000 l-lysine). The method could be applied to molecular epidemiological studies to investigate possible associations between the NEL levels in human tissue proteins and human diseases associated with exposure to AA and alcohol.

Effect of Tetrabutylammonium Cation on Solid-Phase Analytical Derivatization as a Function of Analyte Lipophilicity

Analytical derivatizations (AD) can increase the sensitivity of analyses—including those with mass spectrometric detection—by as much as three orders of magnitude. The extra steps required, however, are a possible impediment to their use. To simplify AD we investigated solid-phase analytical derivatization (SPAD) of compounds with diverse structures by using pentafluorobenzyl bromide (PFBBr) as the reagent. Model compounds were organic acids (e.g. phenols, chlorophenols and carboxylic acids) which were simultaneously extracted and derivatized from 0.1 M NaOH onto a polystyrene–divinylbenzene resin (XAD-4) as their pentafluorobenzyl (PFB) derivatives. Test analytes ranged in molecular weight from 94 for phenol to 266 for pentachlorophenol and octanol–water partition coefficients (log P) values ranged from 1.48 for phenol to 7.15 for hexadecanoic acid. Under SPAD conditions, reaction rates rapidly increased with log P, but yields for less lipophilic compounds, although precise, were unacceptably low. Use of the tetrabutylammonium (TBA) cation as a phase transfer catalyst increased the yield of compounds with low log P; but, unexpectedly, as the log P of the analyte increased, the phase transfer catalyst caused a decrease in yield. The data from this study define the log P range of compounds that require TBA for optimal yield and the log P range of compounds for which TBA compromises yield. This insight led to a simple, two-step, one-pot technique that gave high yields of the PFB derivatives for the entire range of analytes studied. SPAD first extracted/derivatized the lipophilic analytes from aqueous solution onto the solid phase. Extraction/derivatization of the polar analytes followed upon addition of TBA to the reaction mixture. The PFB ethers and esters of the entire range of analytes were then eluted from the XAD-4. The two-step procedure was faster, used less reagent and required lower temperature than comparable methods in the literature. With the two-step procedure, pentafluorobenzylation of phenols and carboxylic acids from water gave yields in excess of 88% with the exception of phenol and pentachlorophenol which were recovered in 57 and 44%, respectively. For all analytes, relative standard deviations were below 15%. The effect of matrix on yield varied between zero and a decline in recoveries of approximately 20–30% depending on the analyte and concentrations of NaCl or humic acid. In the presence of matrix components relative standard deviations remained below 20%.