Erik Hanff

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.

Discovery and microassay of a nitrite-dependent carbonic anhydrase activity by stable-isotope dilution gas chromatography-mass spectrometry.

The intrinsic activity of carbonic anhydrase (CA) is the hydration of CO2 to carbonic acid and its dehydration to CO2. CA may also function as esterase and phosphatase. Recently, we demonstrated that renal CA is mainly responsible for the reabsorption of nitrite (NO2−) which is the most abundant reservoir of the biologically highly potent nitric oxide (NO). By means of a stable-isotope dilution GC–MS method, we discovered a novel CA activity which strictly depends upon nitrite. We found that bovine erythrocytic CAII (beCAII) catalyses the incorporation of 18O from H218O into nitrite at pH 7.4. After derivatization with pentafluorobenzyl bromide, gas chromatographic separation and mass spectrometric analysis, we detected ions at m/z 48 for singly 18O-labelled nitrite (16O=N–18O−/18O=N–16O−) and at m/z 50 for doubly 18O-labelled nitrite (18O=N–18O−) in addition to m/z 46 for unlabelled nitrite. Using 15N-labelled nitrite (15NO2−, m/z 47) as an internal standard and selected-ion monitoring of m/z 46, m/z 48, m/z 50 and m/z 47, we developed a GC–MS microassay for the quantitative determination of the nitrite-dependent beCAII activity. The CA inhibitors acetazolamide and FC5 207A did not alter beCAII-catalysed formation of singly and doubly 18O-labelled nitrite. Cysteine and the experimental CA inhibitor DIDS (a diisothiocyanate) increased several fold the beCAII-catalysed formation of the 18O-labelled nitrite species. Cysteine, acetazolamide, FC5 207A, and DIDS by themselves had no effect on the incorporation of 18O from H218O into nitrite. We conclude that erythrocytic CA possesses a nitrite-dependent activity which can only be detected when nitrite is used as the substrate and the reaction is performed in buffers of neutral pH values prepared in H218O. This novel CA activity, i.e., the nitrous acid anhydrase activity, represents a bioactivation of nitrite and may have both beneficial (via S-nitrosylation and subsequent NO release) and possibly adverse (via C- and N-nitrosylation) effects in living organisms.

Stable-isotope dilution LC-MS/MS measurement of nitrite in human plasma after its conversion to S-nitrosoglutathione

A specific, sensitive and fast LC-MS/MS method with positive electrospray ionization for the quantitative determination of nitrite in human plasma is reported. Added [(15)N]nitrite served as the internal standard (IS). Endogenous nitrite and IS were converted to their S-nitrosoglutathione (GSNO) derivatives, i.e., GS(14)NO and GS(15)NO, respectively, by using excess glutathione (GSH) and HCl. For plasmatic nitrite, fresh plasma (0.5 mL) was spiked with the IS (1000 nM) and ultrafiltered (cut-off 10 kDa). Ultrafiltrate aliquots (100 μL) were treated with aqueous GSH at a final concentration of 1 mM and 1 μL of 5M HCl for 5 min. After final sample dilution (1:1, v/v) with acetonitrile-water (70:30, v/v), 2 μL aliquots were injected via a thermostated (4 °C) autosampler. The mobile phase was acetonitrile-water (70:30, v/v), contained 20mM ammonium formate, had a pH value of 7, and was pumped isocratically at 0.5 mL/min. A Nucleoshell column was used for LC separation. The retention time of GSNO was about 0.8 min and the total analysis time 5 min. Quantification was performed by selected-reaction monitoring the specific mass transition m/z337([M+H](+))→m/z 307([M+H-(14)NO](+·)) for GS(14)NO (i.e., for endogenous nitrite) and m/z338([M+H](+))→m/z307([M+H-(15)NO](+·)) for GS(15)NO (i.e., for the IS). The method was thoroughly validated in human plasma (range, 0-2000 nM). The LOD and LOQ values of the LC-MS/MS method were determined to be 1 fmol and 5 nM [(15)N]nitrite, respectively. The relative matrix-effect of about 21% was outweighed entirely by the IS. In freshly prepared plasma samples from heparinized blood donated by three healthy subjects, nitrite concentration was determined by LC-MS/MS to be 516, 199 and 369 nM. These concentrations were confirmed by using a previously reported GC-MS method and agree with those measured previously by HPLC-UV (334 nm) after nitrite conversion to S-nitroso-N-acetylcysteine (SNAC) by N-acetylcysteine (NAC). Measurement of nitrite by LC-MS/MS as GSNO is about 1000 times more sensitive than by HPLC-UV as SNAC. The applicability of the method to microdialysate, urine, and saliva samples from humans was demonstrated. The agreement of two orthogonal MS-based methods indicates that the concentration of nitrite in freshly prepared, non-frozen plasma from heparinized blood of fasted healthy humans is of the order of 400 nM.

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–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.

Gas chromatographic–mass spectrometric analysis of the tripeptide glutathione in the electron-capture negative-ion chemical ionization mode

The dicarboxylic tripeptide glutathione (GSH) is the most abundant intracellular thiol. GSH analysis by liquid chromatography is routine. Yet, GSH analysis by gas chromatography is challenged due to thermal instability and lacking volatility. We report a high-yield laboratory method for the preparation of 2H-labeled GSH dimethyl ester ((d3Me)2-GSH) for use as internal standard (IS) which was characterized by LC–MS/MS. For GC–MS analysis, the dimethyl esters of GSH and the IS were derivatized with pentafluoropropionic (PFP) anhydride. Electron-capture negative-ion chemical ionization of the (Me)2-(PFP)3-GSH provided high sensitivity. We encourage increasing use of GC–MS in the analysis of amino acids as their Me-PFP derivatives in the ECNICI mode.

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.

Analytical challenges in the assessment of NO synthesis from L-arginine in the MELAS syndrome.

Article history: Received 29 November 2016 Accepted 16 December 2016 Available online xxxx and of de novo synthesized N2-ADMA. Args compartmentalization [5] and concomitant conversion of infused N2-Arg to L-N2homoarginine and N2-guanidinoacetate alter Args metabolism and leads to variable plasma steady states, e.g., for N2-Arg and [N]nitrite/[N]nitrate. Secondly, GC-MSmeasurement of N-enrichmenent in [N]nitrite/

Lacking linearity in quantitative stable-isotope LC–MS/MS measurement of F2-isoprostanes is an irrefutable indicator of analytical inadequacy

In LC-MS/MS-basedquantitative analysis of biological substances such as the F2-isoprostanes including 8-iso-prostaglandin F2α (i.e., 8-iso-PGF2α) use of stable-isotope labelled analogues at matrix-relevant concentrations is indispensable, enables linearity in relevant concentration ranges, and minimizes matrix effects. The quality of the linearity of calibration curves is an indicator of the analytical reliability of the LC-MS/MS method. Poor linearity is a convincing evidence of lacking analytical reliability. Improvement of linearity is better attempted by improving the extraction of the analytes from their biological samples and/or their chromatographic separation accepting longer analysis time, rather than by constructing calibration curves using linear weighted least-squares or other mathematical means.

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.