Pentafluorobenzyl Bromide Research Articles

Twisted Amide-Mediated Peptide Synthesis.

A robust, practical, and sustainable isomerization-suppressed peptide bond formation via acyl sulfonamide, a twisted amide, is disclosed. Tosyl isocyanate and pentafluorobenzyl bromide were applied in combination to activate the peptide C-terminus, which then reacted with an amine to yield an elongated peptide with high stereochemical purity. Careful analysis of NMR spectra of the active intermediate revealed the presence of an intramolecular hydrogen bond, suggesting that the hydrogen bond suppressed Cα-epimerization during amidation. The isomerization suppression by the intramolecular hydrogen bond is expected to be effective even under high dilution conditions, making the present method a powerful tool for the synthesis of complex macrocyclic peptides. In addition to peptide synthesis, the developed synthetic entry to twisted amides can be applied to the investigation of transition metal-catalyzed N-C bond activation. Moreover, the application to the N-C bond activation returned insight into peptide synthesis, leading to the use of sulfonamide as a protecting group of carboxylic acid that can be orthogonally removed in the presence of other conventional protecting groups.

A complementary method with PFBBr-derivatization based on a GC-EI-MS platform for the simultaneous quantitation of short-, medium- and long-chain fatty acids in murine plasma and feces samples.

Fatty acids (FAs) are essential molecules in all organisms and are involved in various physiological and pathophysiological processes. Pentafluorobenzyl bromide (PFBBr) is commonly used for FA derivatization for gas chromatography-mass spectrometry (GC-MS) quantification by chemical ionization (CI). While CI is the conventional ionization mode for PFBBr derivatization, the electron ionization (EI) source has also demonstrated efficacy in achieving satisfactory analytical performance for the analysis of PFB esters. In this study, we present a novel approach utilizing PFBBr-derivatization on a GC-EI-MS platform to quantitatively analyze a comprehensive range of 44 fatty acids (FAs) spanning from C2 to C24. The method's sensitivity, precision, accuracy, linearity, recovery, and matrix effect were rigorously validated against predetermined acceptance criteria. In comparison to the conventional CI ionization mode, the utilization of PFBBr-derivatization in GC-EI-MS exhibits a wider range of applications and achieves comparable sensitivity levels to the conventional CI platform. By using this method, we successfully quantified 44 FAs in plasma and feces samples from the mice with deoxynivalenol (DON)-induced kidney injury. Among these, the levels of most FA species were increased in the DON-exposure group compared with the control group. The orthogonal partial least squares discriminant analysis (OPLS-DA) of all the tested FAs showed a visual separation of the two groups, indicating DON exposure resulted in a disturbance of the FA profile in mice. These results indicate that the established method by integration of GC-MS with PFBBr derivatization is an efficient approach to quantify the comprehensive FA profile, which includes short-, medium- and long-chain FAs. In addition, our study provides new insights into the mechanism underlying DON exposure-induced kidney injury.

The stability of cyanide in human biological samples. A systematic review, meta-analysis and determination of cyanide (GC-QqQ-MS/MS) in an authentic casework 7 years after fatal intoxication

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.

GC/EI-MS Analysis of Amino Acids Derivatized with Pentafluorobenzyl Bromide

GC/EI-MS Analysis of Amino Acids Derivatized with Pentafluorobenzyl Bromide

Detection of thiocyanate in postmortem blood of a fatal potassium thiocyanate-poisoning patient using gas chromatography-mass spectrometry.

Thiocyanate is an inorganic compound used in industrial applications. Here, we report a case of suicidal death due to acute thiocyanate overdose. A 44-year-old man who consumed an unknown amount of thiocyanate solution was transferred to the emergency room and died two hours after admission. An autopsy was performed two days after death. General toxicological analysis of blood using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry found no drug or alcohol. Quantification using GC-MS post-derivatization with pentafluorobenzyl bromide revealed 2,290 and 1,920 mg/L of thiocyanate in the heart and femoral blood samples, respectively. Thus, the cause of death was attributed to thiocyanate overdose. This study provides useful information for the interpretation of thiocyanate-related fatalities.

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.

HiSorb sorptive extraction for determining salivary short chain fatty acids and hydroxy acids in heart failure patients

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.

GC-MS Studies on Nitric Oxide Autoxidation and S-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using 15N and 18O Isotopes.

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.

UiO-66-NH2: a recyclable and efficient sorbent for dispersive solid-phase extraction of fluorinated aromatic carboxylic acids from aqueous matrices.

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 beprecise, reproducible, and applicable to the seawater samples. In the linearity range, the regression value was foundto be >0.98; LOD and LOQ were foundto be in the range of0.33-1.17ng/mL and 1.23-3.33ng/mL, respectively; and the value of theextraction 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 amaximum RSD value of6.87% that confirms the applicability of the method to different water matrices.

Comparison of Low-Density Lipoprotein Oxidation by Hydrophilic O(3 P)-Precursors and Lipid-O(3 P)-Precursor Conjugates.

Lipid oxidation by reactive oxygen species (ROS) provide several different oxidation products that have been implicated in inflammatory responses. Ground state atomic oxygen [O(3 P)] is produced by the photodeoxygenation of certain heterocyclic oxides and has a reactivity that is unique from other ROS. Due to the reactive nature of O(3 P), the site of O(3 P)-generation is expected to influence the products in heterogenous solutions or environments. In this work, the oxidation of low-density lipoprotein (LDL) by lipids with covalently bound O(3 P)-photoprecursors was compared to more hydrophilic O(3 P)-photoprecursors. Lipid oxidation products were quantified after Bligh-Dyer extraction and pentafluorobenzyl bromide (PFB) derivatization by GC-MS. Unlike the more hydrophilic O(3 P)-photoprecursors, the oxidation of LDL during the irradiation of lipid-(O3 P)-photoprecursor conjugates showed little quenching by the addition of the O(3 P)-scavenging sodium allyl sulfonate. This indicated that lipophilic O(3 P)-photoprecursors are expected to generate lipid oxidation products where other more hydrophilic O(3 P)-photoprecursors could be quenched by other reactive groups present in solution or the environment.

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.

Unusual Derivatization of Methylmalonic Acid with Pentafluorobenzyl Bromide to a Tripentafluorobenzyl Derivative and Its Stable-Isotope Dilution GC-MS Measurement in Human Urine.

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.

Suicide by sodium nitrite–an emerging trend

Suicide by sodium nitrite ingestion is increasingly being reported in many parts of the world due to the ease of access of information and material via the internet. Our laboratory received our first case of suspected nitrite fatality in October 2021, and soon after, a second case in November 2021. Both deceased were found dead at their home with packet/bottle of sodium nitrite present in their rooms. The deceased in the first case was a 29-year-old female and the specimens submitted for toxicology analyses were peripheral blood, bile, vitreous humour and gastric contents. The second case was a 16-year-old male and his peripheral blood and urine were submitted for toxicology analyses. To prove nitrite consumption, a relatively simple colorimetric method using commercially available Griess reagent was evaluated in the vitreous humour and gastric contents in case 1 and urine in case 2. The Griess assay was adopted from the work of Tomsia M. et. al (J. Forensic. Leg. Med., 2021, 102186). It involves the reaction between N-(1-naphtyl)ethylenediamine dichloride and nitrite to form a diazonium salt which further react with sulphanilamide contained in the Griess reagent to produce a purple coloured azo compound. The colour of the azo compound increases proportionally with nitrite concentration and can be measured by a UV-Vis spectrophotometer at around 548 nm. For quantitation, calibration curves from 0.5 to 10 μg/ml nitrite were prepared in the respective matrices (vitreous humour, urine and water for gastric contents). To further confirm the presence of nitrite in these specimens, a complementary technique by GC/MS using pentafluorobenzyl bromide (PFB-Br) derivatization is employed (Tsikas D., J. Chromatogr. B, 2016, 1043:187-201). Nitrite levels determined by Griess assay were found to be 78 μg/ml in the vitreous humour and 32 mg/ml in the gastric contents of case 1, and 13.9 μg/ml in the urine of case 2. These levels were comparable to those reported by Tomsia M et. al. The nitrite levels in the vitreous humour and urine were also far above the typical endogenous nitrite levels (around 0.01–0.3 μg/ml in vitreous humour and 0.01–0.05 μg/ml in urine). The presence of nitrite as α-nitro-pentafluorotoluene was also confirmed in the 3 specimens by GC/MS. Nitrate does not interfere in both the Griess assay and GC/MS method. Blood nitrite levels in nitrite poisoning cases were often present at levels not significantly different from that of endogenous levels as it is rapidly oxidized to nitrate by haemoglobin. For this reason, blood nitrate levels were determined instead in some of the reported nitrite ingestion cases. However, the determination of nitrite in the deceased's specimen is still preferred as a direct correlation to the substance of poisoning. In this aspect, vitreous humour is a useful specimen as it is relatively clean and the easiest to process. This case study presented the use of Griess reagent and PFB-Br derivation with GC/MS analysis for the quantitation and confirmation of nitrite in vitreous humour, gastric contents and urine specimens of nitrite ingestion cases. The approach is simple and employs relatively inexpensive reagents and instruments that are readily available in most toxicology laboratories.

A case series of suicides by sodium azide ingestion

To investigate the involvement of sodium azide in 13 forensic apparent suicides. Cases with suspicion of an intentional sodium azide ingestion confirmed by blood analysis were included. All blood samples were collected at the scene by the forensic physician. In six cases, the blood was femoral blood, in the other cases the origin was unknown. Nine blood samples were preserved with sodium fluoride and heparin or oxalate as anticoagulant, four contained EDTA as anticoagulant. The cases were screened for the presence of alcohol, drugs of abuse and medicines in blood. Azide in blood was identified and quantified after derivatization with pentafluorobenzylbromide (PFBBr) using GC-MS. In some cases other biological matrices were available for analysis as well. Thirteen cases were included (male/female: 2/11; age range: 27–86 years). Most bodies were found in their homes. Two victims were found in the garden needing resuscitation and eventually died in the hospital. In almost all cases, a small container labeled with sodium azide was found at the scene and the total dose in the container, if classified, varied from 2 to 3 gram. In three cases, a forensic autopsy was performed and the pathologist found no anatomical cause of death. Toxicological analysis for the presence of alcohol, drugs of abuse and medicines resulted in no indications for a toxicological cause of death. In all cases, medicines were found, especially analgesics, opioids and anti-emetics. Alcohol was detected in three cases. The median azide blood concentration was 17 mg/L (interquartile range: 13.5–20.5 mg/L). Azide was measured in other biological matrices in four cases showing higher azide concentrations in vitreous humor and lower azide concentrations in heart blood and urine compared to the blood obtained by the forensic physician. Median concentrations in vitreous humor, heart blood and urine were 26, 8 and 7 mg/L, respectively. In 11 cases, very high concentrations of azide were found in the blood (13 to 26 mg/L), corresponding with concentrations that were measured in persons who died after apparent intentional ingestion of sodium azide. In two cases, no toxicologically relevant results were found, except for azide. However, the measured azide concentrations were lower (about 1 mg/L), possibly due to degradation as a consequence of prolonged unsuitable storage prior to arrival at the institute. Unfortunately, no other biological matrices were available. These victims were found together and a container with label of sodium azide was found near them. The mentioned dose on the label of 3 gram is higher than earlier reported lethal doses of 700 mg. Higher azide concentrations in vitreous humor suggest good permeation and postmortem stability. It was concluded that the high concentration of azide can explain the death in 11 of our cases. In 2 cases, death by an azide overdose cannot be concluded but also cannot be excluded. Although femoral blood is the specimen of choice, vitreous humor is a good alternative when suspecting an azide overdose.

Potential of atmospheric pressure ionization sources for the analysis of free fatty acids in clinical and biological samples by gas chromatography-mass spectrometry

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.

Quantitation of Fatty Acids in Serum/Plasma and Red Blood Cells by Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry.

Quantitation of long-chain fatty acids in serum/plasma and red blood cells is a useful diagnostic tool in the evaluation of nutritional status and assessment of risk for essential fatty acid deficiency (EFAD). Serum/plasma has been the traditional sample type for this method, yet it requires prolonged fasting which is not compatible with some patient populations. More recently, red blood cells have become an important sample type due to less intraindividual variability and obviating the need for fasting. Here we present a method for the quantitation of 22 fatty acids in serum/plasma or red blood cells. Fatty acids are hydrolyzed and extracted from the biological matrix, followed by derivatization with pentafluorobenzyl bromide and subsequent analysis by gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS).

GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization.

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.

Determination of Cyanide in Blood for Forensic Toxicology Purposes-A Novel Nci Gc-Ms/Ms Technique.

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.

Determination of Methoxyacetic acid in urine by pre-column derivatization-liquid-liquid microextraction coupled with gas chromatography

Objective: To establish a method for determining methoxyacetic acid in urine by pre-column derivatization-liquid-liquid microextraction coupled with gas chromatography (GC) . Methods: Phosphate buffer solution, tert-butoxyacetic acid (internal standard) and pentafluorobenzyl bromide (derivative) were added to the urine sample. After derived in a water bath at 90 ℃ for 40 min, the mixture was cooled and filtered, then the dichloromethane was used as an extractant. After being shaken and centrifuged, the lower organic phase was sucked and injected into a gas chromatograph, separated by a DB-5 capillary column, and detected by an ECD detector. Results: The linear range of the method was 0.6~60.0 mg/L with the correlation coefficients (r) above 0.999. The average recovery was76.6%~110.7%, the inter-day precision was 8.00%~8.82%, and the detection limit was 0.13 mg/L. Conclusion: The method was founded to be high sensitivity, low organic reagent usage and green. So it is suitable for the detection of methoxyacetic acid in urine of occupational exposure to ethylene glycol monomethyl ether.

Determination of Azide Ions in Blood by Pentafluorobenzyl Derivation Followed by GC-MS.

Objective To establish a method for determination of the azide ions in blood by gas chromatography-mass spectrometry （GC-MS） following pentafluorobenzyl derivatization. Methods A blood sample of 0.2 mL was placed into a 10 mL glass test tube, and the internal standard sodium cyanide, derivatization reagent pentafluorobenzyl bromide and catalyst tetradecyl benzyl dimethyl ammonium chloride were added in turn. After vortex mixing, the mixture was heated with low-power microwave for 3 min. After centrifugation, the organic phase was taken for GC-MS analysis. Results The azide ions in blood had a good linear relationship in the mass concentration range of 0.5 to 20 μg/mL. The lowest detection limit was 0.25 μg/mL and the relative recovery was 91.36%-94.58%. The method was successfully applied to a case of death from sodium azide poisoning. The mass concentration of azide ions in the blood of the dead was 11.11 μg/mL. Conclusion The method developed in this paper has strong specificity and is easy to operate, which is suitable for the rapid detection of azide ions in blood.