A 30 year old man was found with no signs of life in front of the house. The cyanide concentration in blood and urine was determined five years after the man\'s death. What is more, a stability study was conducted for 730 days in an authentic casework blood sample. Sample preparation procedure included precipitation with methanol:water mixture, solid phase extraction (SPE) and derivatization with the use of PFB-Br (pentafluorobenzyl bromide). The sample was analyzed using GC-QqQ-MS/MS (gas chromatopraphy coupled with tandem mass spectrometry) isotope dilution method. Separation was done using a SH-RXI-5MS column (30 m x 0.25 mm, 0.25 µm). Detection of PFB-CN and PFB-13CN was achieved using a triple-quadrupole mass spectrometer with an electron ionization (EI) ion source in multiple reaction monitoring (MRM) mode. After 5 years from the man\'s death, cyanide concentration was: 1900 ng/mL in blood and 500 ng/mL in urine. Stability study performed in an authentic blood sample 6 and 7 years after the man\'s death revealed cyanide concentrations of 1898.2 ng/mL and 1618.7 ng/mL, respectively. While spectrophotometric and colorimetric methods recorded both decrease and increase in cyanide concentration over time, newer chromatographic methods mainly indicate a decrease. The studies presented in this paper seem to confirm this trend. However, in order to interpretate the results of cyanide concentration in biological material reliably, more research is still necessary.

Variations in salivary short-chain fatty acids and hydroxy acids (e.g., lactic acid, and 3-hydroxybutyric acid) levels have been suggested to reflect the dysbiosis of human gut microbiota, which represents an additional factor involved in the onset of heart failure (HF) disease. The physical-chemical properties of these metabolites combined with the complex composition of biological matrices mean that sample pre-treatment procedures are almost unavoidable. This work describes a reliable, simple, and organic solvent free protocol for determining short-chain fatty acids and hydroxy acids in stimulated saliva samples collected from heart failure, obese, and hypertensive patients. The procedure is based on in-situ pentafluorobenzyl bromide (PFB-Br) derivatization and HiSorb sorptive extraction coupled to thermal desorption and gas chromatography-tandem mass spectrometry. The HiSorb extraction device is completely compatible with aqueous matrices, thus saving on time and materials associated with organic solvent-extraction methods. A Central Composite Face-Centred experimental design was used for the optimization of the molar ratio between PFB-Br and target analytes, the derivatization temperature, and the reaction time which were 100, 60 °C, and 180 min, respectively. Detection limits in the range 0.1-100 µM were reached using a small amount of saliva (20 µL). The use of sodium acetate-1-13C as an internal standard improved the intra- and inter-day precision of the method which ranged from 10 to 23%. The optimized protocol was successfully applied for what we believe is the first time to evaluate the salivary levels of short chain fatty acids and hydroxy acids in saliva samples of four groups of patients: i) patients admitted to hospital with acute HF symptoms, ii) patients with chronic HF symptoms, iii) patients without HF symptoms but with obesity, and iv) patients without HF symptoms but with hypertension. The first group of patients showed significantly higher levels of salivary acetic acid and lactic acid at hospital admission as well as the lowest values of hexanoic acid and heptanoic acid. Moreover, the significant high levels of acetic acid, propionic acid, and butyric acid observed in HF respect to the other patients suggest the potential link between oral bacteria and gut dysbiosis.

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+; pKaHONO, 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.

The present study describes the trace analysis of 23 fluorinated aromatic carboxylic acids based on the dispersive solid-phase extraction (dSPE) technique using UiO-66-NH2 MOF as efficient, recyclable sorbent, and GC-MS negative ionization mass spectrometry (NICI MS) as determination technique. All 23 fluorobenzoic acids (FBAs) were enriched, separated, and eluted in a shorter retention time; the derivatization was done by pentafluorobenzyl bromide (1% in acetone), in which the use of inorganic base K2CO3 was improved by triethylamine to increase the lifespan of the GC column. The performance of UiO-66-NH2 was evaluated by dSPE in Milli-Q water, artificial seawater, and tap water samples, and the impact of various parameters on the extraction efficiency was investigated by GC-NICI MS. The method was found to be precise, reproducible, and applicable to the seawater samples. In the linearity range, the regression value was found to be >0.98; LOD and LOQ were found to be in the range of 0.33-1.17 ng/mL and 1.23-3.33 ng/mL, respectively; and the value of the extraction efficiency was found to range between 98.45 and 104.39% for Milli-Q water samples, 69.13-105.48% for salt-rich seawater samples, and 92.56-103.50% for tap water samples with a maximum RSD value of 6.87% that confirms the applicability of the method to different water matrices.

Methylmalonic acid (MMA) is a very short dicarboxylic acid (methylpropanedioic acid; CH3CH(COOH)2; pKa1, 3.07; pKa2, 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.

Because of the central role of fatty acids in biological systems, their accurate quantification is still important. However, the impact of the complex matrix of biologically and clinically relevant samples such as plasma, serum, or cells makes the analysis still challenging, especially, when free non-esterified fatty acids have to be quantified. Here we developed and characterized a novel GC-MS method using pentafluorobenzyl bromide as a derivatization agent and compared different ionization techniques such as atmospheric pressure chemical ionization (APCI), atmospheric pressure chemical photoionization (APPI), and negative ion chemical ionization (NICI). The GC-APCI-MS showed the lowest limits of detection from 30 to 300 nM for a broad range of fatty acids and a similar response for various fatty acids from a chain length of 10 to 20 carbon atoms. This allows the number of internal standards necessary for accurate quantification to be reduced. Moreover, the use of pentafluorobenzyl bromide allows the direct derivatization of free fatty acids making them accessible for GC-MS analysis without labor-intense sample pretreatment.

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.

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.

The high toxicity of cyanide, along with its widespread industrial use, has fuelled interest in the development of analytical methods for its determination in complex matrices. In this study, we propose a novel approach for the measurement of total cyanide in soil samples based on single-step derivatization with pentafluorobenzyl bromide (F5Bn-Br) followed by quantitation with gas chromatography mass spectrometry in negative chemical ionization mode. The reaction between CN- and F5Bn-Br resulted in the identification of several derivatives such as F5Bn-CN, (F5Bn)(F5Ph)CH-CN, and (F5Bn)2(F5Ph)C-CN. The relative proportion between such compounds was dependent on experimental conditions. When a 100 μL aliquot of an alkaline-aqueous extract was reacted with 700 μL of 1.3% F5Bn-Br in acetone, the tri-alkylated derivative was the most abundant. In such conditions a detection limit of 0.5 ng/g of CN- was attained. Soil samples were initially spiked with an alkaline solution of K13C15N internal standard and suspended in 7.5% aqueous NaOH. Determination of total cyanide was achieved by digestion of the alkaline extract with H3PO4 to produce HCN which was then trapped in 0.1% NaOH in a sealed double vial system, followed by reaction with F5Bn-Br. Isotope dilution calibration was chosen for quantitation, and the validity of the novel method was demonstrated by analysis of soil Certified Reference Materials (CRMs) and by spike recovery tests.

Methylisothiazolinone and the mixture of chloromethylisothiazolinone/methylisothiazolinone (MCI/MI, 3:1) are widespread biocides used in cosmetic and household products. Due to their skin permeability, they might be taken up by the general population via use of products containing these biocides. As both compounds are known skin sensitizers, the use of these products is under discussion by regulatory agencies. In order to evaluate the possible uptake of MI and/or MCI/MI by human biomonitoring, we have developed and validated a highly sensitive and specific GC/MS/MS-method for the quantification of N-methylmalonamic acid (NMMA), a known metabolite of MI and MCI in urine of rats. After freeze-drying of urine, the analyte is derivatised with pentafluorobenzyl bromide in anhydrous solution and the PFB-derivative is extracted into n-hexane. After concentration, the derivative is finally quantified by GC/MS/MS in EI-mode using 13C3-NMMA as internal standard. The limit of quantification for NMMA was 0.5ngmL-1 urine. Precision within and between-series was determined to range between 3.7-10.9% using native and spiked quality control samples. Accuracy ranged between 89 and 114%. In a pilot study we applied this method to spot urine samples of 63 persons not knowingly exposed to MI and/or MCI/MI. NMMA was quantifiable in every urine sample analysed, with no significant difference in urinary levels between male and female participants. The median (95th percentile) levels for urinary NMMA were 3.6 (7.4) ngmg-1 creatinine and 2.9 (9.1) ngmg-1 creatinine for males (n=32) and females (n=31), respectively. In a volunteer experiment, a relation of exposure to MI and/or MCI/MI and subsequent NMMA-excretion was shown. Our method is the first to report human urinary background levels of NMMA. However, the possibility of formation and urinary excretion of NMMA within physiological processes cannot be ruled out.