Search Results (8)

Nitrite (O=N-O⁻, NO₂⁻) and nitrate (O=N(O)-O⁻, NO₃⁻) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (^●NO). ^●NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of ^●NO synthases. It is considered that the autoxidation of ^●NO in aqueous solutions and in O₂-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO^●) intermediates. In aqueous buffers, endogenous S-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., S-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., S-nitrosoglutathione, GSNO) may be formed during the autoxidation of ^●NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O⁻ + H⁺; pK_a^HONO, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of ^●NO. This work describes in vitro GC-MS studies on the reactions of unlabeled (¹⁴NO₂⁻) and labeled nitrite (¹⁵NO₂⁻) and RSNO (RS¹⁵NO, RS¹⁵N¹⁸O) performed in pH-neutral aqueous buffers of phosphate or tris(hydroxyethylamine) prepared in unlabeled (H₂¹⁶O) or labeled H₂O (H₂¹⁸O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography–mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of ^●NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl₂ accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating ¹⁸O from H₂¹⁸O into the SNO group. In aqueous buffers prepared in H₂¹⁸O, synthetic peroxynitrite (ONOO⁻) decomposes to nitrite without ¹⁸O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS¹⁵NO and H₂¹⁸O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of ^●NO and hydrolysis of RSNO. Full article

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. Full article

Methylmalonic acid (MMA) is a very short dicarboxylic acid (methylpropanedioic acid; CH₃CH(COOH)₂; pK_a1, 3.07; pK_a2, 5.76) associated with vitamin B₁₂ deficiency and many other patho-physiological conditions. In this work, we investigated several carboxylic groups-specific derivatization reactions and tested their utility for the quantitative analysis of MMA in human urine and plasma by gas chromatography-mass spectrometry (GC-MS). The most useful derivatization procedure was the reaction of unlabeled MMA (d₀-MMA) and trideutero-methyl malonic acid (d₃-MMA) with 2,3,4,5,6-pentafluorobenzyl bromide (PFB-Br) in acetone. By heating at 80 °C for 60 min, we observed the formation of the dipentafluorobenzyl (PFB) ester of MMA (CH₃CH(COOPFB)₂). In the presence of N,N-diisopropylamine, heating at 80 °C for 60 min resulted in the formation of a tripentafluorobenzyl derivative of MMA, i.e., CH₃CPFB(COOPFB)₂). The retention time was 5.6 min for CH₃CH(COOPFB)₂ and 7.3 min for CH₃CPFB(COOPFB)₂). The most intense ions in the negative-ion chemical ionization (NICI) GC-MS spectra of CH₃CH(COOPFB)₂ were mass-to-charge (m/z) 233 for d₀-MMA and m/z 236 for d₃-MMA. The most intense ions in the NICI GC-MS spectra of CH₃CPFB(COOPFB)₂ were mass-to-charge (m/z) 349 for d₀-MMA and m/z 352 for d₃-MMA. These results indicate that the H at C atom at position 2 is C-H acidic and is alkylated by PFB-Br only in the presence of the base N,N-diisopropylamine. Method validation and quantitative analyses in human urine and plasma were performed by selected ion monitoring (SIM) of m/z 349 for d₀-MMA and m/z 352 for the internal standard d₃-MMA in the NICI mode. We used the method to measure the urinary excretion rates of MMA in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). The creatinine-corrected excretion rates of MMA were 1.50 [0.85–2.52] µmol/mmol in the black boys and 1.34 [1.02–2.18] µmol/mmol in the white boys (P = 0.85; Mann–Whitney). The derivatization procedure is highly specific and sensitive for MMA and allows its accurate and precise measurement in 10-µl of human urine by GC-MS. Full article

Carbon dioxide (CO₂) and carbonates, which are widely distributed in nature, are constituents of inorganic and organic matter and are essential in vegetable and animal organisms. CO₂ is the principal greenhouse gas in the atmosphere. In human blood, CO₂/HCO₃⁻ is an important buffering system. Inorganic nitrate (ONO₂⁻) and nitrite (ONO⁻) are major metabolites and abundant reservoirs of nitric oxide (NO), an endogenous multifunctional signaling molecule. Carbonic anhydrase (CA) is involved in the reabsorption of nitrite and nitrate from the primary urine. The measurement of nitrate and nitrite in biological samples is of particular importance. The derivatization of nitrate and nitrite in biological samples alongside their ¹⁵N-labeled analogs, which serve as internal standards, is a prerequisite for their analysis by gas chromatography–mass spectrometry (GC-MS). A suitable derivatization reagent is pentafluorobenzyl bromide (PFB-Br). Nitrate and nitrite are converted in aqueous acetone to PFB-ONO₂ and PFB-NO₂, respectively. PFB-Br is also useful for the GC-MS analysis of carbonate/bicarbonate. This is of particular importance in conditions of pharmacological CA inhibition, for instance by acetazolamide, which is accompanied by elevated concomitant excretion of nitrate, nitrite and bicarbonate, as well as by urine alkalization. We performed a series of experiments with exogenous bicarbonate (NaHCO₃) added to human urine samples (range, 0 to 100 mM), as well as with endogenous bicarbonate resulting from the inhibition of CA activity in healthy subjects before and after ingestion of pharmacological acetazolamide. Our results indicate that bicarbonate enhances the derivatization of nitrate with PFB-Br. In contrast, bicarbonate decreases the derivatization of nitrite with PFB-Br. Bicarbonate is not a catalyst, but it enhances PFB-ONO₂ formation and inhibits PFB-NO₂ formation in a concentration-dependent manner. The effects of bicarbonate are likely to result from its reaction with PFB-Br to generate PFB-OCOOH. Nitrate reacts with concomitantly produced PFB-OCOOH to form PFB-ONO₂ in addition to the direct reaction of nitrate with PFB-Br. By contrast, nitrite does not react with PFB-OCOOH to form PFB-NO₂. Sample acidification by small volumes of 20 wt.% aqueous acetic acid abolishes the effects of exogenous and endogenous bicarbonate on nitrite measurement. Full article

One of the recently evolving methods for cyanide determination in body fluids is GC-MS, following extractive alkylation with pentafluorobenzyl bromide or pentafluorobenzyl p-toluenesulfonate. The aim of this study was to improve previous GC methods by utilizing a triple quadrupole mass spectrometer, which could enhance selectivity and sensitivity allowing for the reliable confirmation of cyanide exposure in toxicological studies. Another purpose of this study was to facilitate a case investigation including a determination of cyanide in blood and to use the obtained data to confirm the ingestion of a substance, found together with a human corpse at the forensic scene. The blood samples were prepared following extractive alkylation with a phase transfer catalyst tetrabutylammonium sulfate and the PFB-Br derivatization agent. Optimal parameters for detection, including ionization type and multiple reaction monitoring (MRM) transitions had been investigated and then selected. The validation parameters for the above method were as follows—linear regression R² = 0.9997 in the range of 0.1 µg/mL to 10 µg/mL; LOD = 24 ng/mL; LOQ = 80 ng/mL and an average recovery of extraction of 98%. Our study demonstrates the first attempt of cyanide determination in blood with gas chromatography-tandem mass spectrometry. The established method could be applied in forensic studies due to MS/MS confirmation of organic cyanide derivative and low matrix interferences owning to utilizing negative chemical ionization. Full article

In consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1-methyl-5H-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to correct the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measurement of creatinine by gas chromatography (GC)-based methods requires derivatization of its amine and keto groups. Creatinine exists in equilibrium with its open form creatine (methylguanidoacetic acid, MW 131.1), which has a guanidine and a carboxylic group. Trimethylsilylation and trifluoroacetylation of creatinine and creatine are the oldest reported derivatization methods for their GC-mass spectrometry (MS) analysis in human serum using flame- or electron-ionization. We performed GC-MS studies on the derivatization of creatinine (d₀-creatinine), [methylo-²H₃]creatinine (d₃-creatinine, internal standard) and creatine (d₀-creatine) with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) using standard derivatization conditions (60 min, 60 °C), yet in the absence of any base. Reaction products were characterized both in the negative-ion chemical ionization (NICI) and in the positive-ion chemical ionization (PICI) mode. Creatinine and creatine reacted with BSTFA to form several derivatives. Their early eluting N,N,O-tris(trimethylsilyl) derivatives (8.9 min) were found to be useful for the precise and accurate measurement of the sum of creatinine and creatine in human urine (10 µL, up to 20 mM) by selected-ion monitoring (SIM) of m/z 271 (d₀-creatinine/d₀-creatine) and m/z 274 (d₃-creatinine) in the NICI mode. In the PICI mode, SIM of m/z 256, m/z 259, m/z 272 and m/z 275 was performed. BSTFA derivatization of d₀-creatine from a freshly prepared solution in distilled water resulted in formation of two lMate-eluting derivatives (14.08 min, 14.72 min), presumably creatinyl-creatinine, with the creatininyl residue existing in its enol form (14.08 min) and keto form (14.72 min). Our results suggest that BSTFA derivatization does not allow specific analysis of creatine and creatinine by GC-MS. Preliminary analyses suggest that pentafluoropropionic anhydride (PFPA) is also not useful for the measurement of creatinine in the presence of creatine. Both BSTFA and PFPA facilitate the conversion of creatine to creatinine. Specific measurement of creatinine in urine is possible by using pentafluorobenzyl bromide in aqueous acetone. Full article

A simple method for tracing carbon fixation and lipid synthesis in microalgae was developed using a combination of solid-phase extraction (SPE) and negative ion chemical ionisation gas chromatography mass spectrometry (NCI-GC-MS). NCI-GC-MS is an extremely sensitive technique that can produce an unfragmented molecular ion making this technique particularly useful for stable isotope enrichment studies. Derivatisation of fatty acids using pentafluorobenzyl bromide (PFBBr) allows the coupling of the high separation efficiency of GC and the measurement of unfragmented molecular ions for each of the fatty acids by single quadrupole MS. The key is that isotope spectra can be measured without interference from co-eluting fatty acids or other molecules. Pre-fractionation of lipid extracts by SPE allows the measurement of ¹³C isotope incorporation into the three main lipid classes (phospholipids, glycolipids, neutral lipids) in microalgae thus allowing the study of complex lipid biochemistry using relatively straightforward analytical technology. The high selectivity of GC is necessary as it allows the collection of mass spectra for individual fatty acids, including cis/trans isomers, of the PFB-derivatised fatty acids. The combination of solid-phase extraction and GC-MS enables the accurate determination of ¹³C incorporation into each lipid pool. Three solvent extraction protocols that are commonly used in lipidomics were also evaluated and are described here with regard to extraction efficiencies for lipid analysis in microalgae. Full article