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-MS/MS measurement of malondialdehyde and 4-hydroxy-2-nonenal in human plasma: Effects of long-term L-arginine administration.

Here, we report the simultaneous derivatization and quantification of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) in human plasma by GC-MS/MS using [1,3-2H2]-MDA (d2-MDA) and [9,9,9-2H3]-HNE (d3-HNE) as the internal standards, respectively. MDA, d2-MDA, HNE and d3-HNE were converted to their pentafluorobenzyl oximes (PFBOX) by pentafluorobenzyl hydroxylamine. Subsequently, the hydroxyl groups of the PFBOX of HNE and d3-HNE were trimethylsilylated with N,O-bis(trimethylsilyl)trifluoroacetamide/1% trimethylchlorosilane. GC-MS/MS analyses were performed in the electron-capture negative-ion chemical ionization mode. Quantification was performed by selected-reaction monitoring the mass transitions m/z 442 to m/z 243 for MDA, m/z 444 to m/z 244 for d2-MDA, m/z 403 → m/z 283 for HNE and m/z 406 → m/z 286 for d3-HNE. The method was applied to measure MDA and HNE in plasma of patients suffering from coronary artery disease (CAD) or peripheral artery occlusive disease (PAOD) before and after oral supplementation of L-arginine (3 g/day) or placebo for 3 (CAD and PAOD) and 6 months (PAOD). All plasma samples were analyzed after completion of the studies. Our results revealed that storage of plasma samples (at -80 °C) leads to lower MDA and HNE plasma concentrations in the plasma samples that were collected at the end of the studies as compared to those collected at the begin of the studies. Based on MDA and HNE measurements in plasma, L-arginine did not influence lipid peroxidation in CAD and PAOD patients. Long-term studies on lipid peroxidation are best performed by measuring oxidative stress biomarkers such as MDA and/or HNE in plasma samples immediately after their collection. Long-term storage of plasma samples even at -80 °C is not recommended.

GC-MS and GC-MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples.

L-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-(15)N2]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC-MS and selected-reaction monitoring in GC-MS/MS. We obtained very similar hArg concentrations by GC-MS and GC-MS/MS, suggesting that GC-MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC-MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.

GC-MS/MS and LC-MS/MS studies on unlabelled and deuterium-labelled oleic acid (C18:1) reactions with peroxynitrite (O=N-O-O⁻) 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.

UPLC-MS/MS measurement of S-nitrosoglutathione (GSNO) in human plasma solves the S-nitrosothiol concentration enigma.

We developed and validated a fast UPLC-MS/MS method with positive electrospray ionization (ESI+) for the quantitative determination of S-nitrosoglutathione (GSNO) in human plasma. We used a published protocol for the inactivation of plasma γ-glutamyltransferase (γGT) activity by using the γGT transition inhibitor serine/borate and the chelator EDTA for the stabilization of GSNO, and N-ethylmaleimide (NEM) to block SH groups and to avoid S-transnitrosylation reactions which may diminish GSNO concentration. S-[(15)N]Nitrosoglutathione (GS(15)NO) served as internal standard. Fresh blood was treated with NEM/serine/borate/EDTA, plasma spiked with GS(15)NO (50nM) was ultrafiltered (cut-off 10kDa) and 10µL aliquots of the ultrafiltrate were analyzed by UPLC-MS/MS. Five HILIC columns and an Acquity UPLC BH amide column were tested. The mobile phase was acetonitrile-water (70:30, v/v), contained 20mM ammonium formate, had a pH value of 7, and was pumped isocratically (0.5mL/min). The Nucleoshell column allowed better LC performance and higher MS sensitivity. The retention time of GSNO was about 1.1min. Quantification was performed by selected-reaction monitoring the mass transition m/z 337 ([M+H](+))→m/z 307 ([M+H(14)NO](+)) for GSNO (i.e., GS(14)NO) and m/z 338 ([M+H](+))→m/z 307 ([M+H(15)NO](+)) for GS(15)NO. NEM/serine/borate/EDTA was found to stabilize GSNO in human plasma. The method was validated in human plasma (range, 0-300nM) using 50nM GS(15)NO. Accuracy and precision were in generally acceptable ranges. A considerable matrix effect was observed, which was however outweighed by the internal standard GS(15)NO. In freshly prepared plasma from heparinized blood donated by 10 healthy subjects, no endogenous GSNO was determined above 2.8nM, the limit of quantitation (LOQ) of the method. This study challenges previously reported GSNO plasma concentrations being far above the present method LOQ value and predicts that the concentration of low-molecular-mass and high-molecular-mass S-nitrosothiols are in the upper pM- and lower nM-range, respectively.

A validated, rapid UPLC-MS/MS method for simultaneous ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples.

A recent clinical trial assessing human autonomic cardiovascular regulation applied pacemaker channel inhibition with ivabradine, norepinephrine transporter blockade with reboxetine, and beta-adrenoreceptor blockade with metoprolol. To verify patient adherence, we developed and validated a fast UPLC-MS/MS assay measuring all three compounds simultaneously. Deuterium-labeled drugs, d3-ivabradine, d5-reboxetine and d7-metoprolol, served as internal standards. Sample preparation of 200µL human plasma consisted of a single liquid-liquid extraction step by means of ethyl acetate. Chromatographic separation was performed on a 50-mm long BEH C18 column with gradient elution using a mixture of water and methanol each containing 2mM ammonium acetate over 4.5min. The mass spectrometer was operated in the positive electrospray ionization (ESI+) mode. Characteristic product ions resulting from collision-induced dissociation of unlabeled and deuterium-labeled drugs with argon were used for quantification in the selected-reaction monitoring mode. We validated the method according to the European Medicines Agency (EMA) guideline on bioanalytical method validation over the range from 1ng/mL to 500ng/mL for all three analytes. Linear responses with correlation coefficients>0.99 over that range were acquired. The LOQ value was 1ng/mL for each drug. Regulatory criteria for accuracy (80-120%) and precision (RSD<15%) were met for all drugs. The internal standard-normalized matrix factor was close to 1 for low and high analyte concentrations. We successfully measured ivabradine, reboxetine, and metoprolol concentrations in 107 human plasma samples from a clinical trial. Quality control samples processed in parallel confirmed the method's reliability in a clinical setting.

Glutathione promotes prostaglandin H synthase (cyclooxygenase)-dependent formation of malondialdehyde and 15(S)-8-iso-prostaglandin F2α.

Prostaglandin (PG) H synthases (PGHS) or cyclooxygenases (COX) catalyse the peroxidation of arachidonic acid (AA) to PGG(2) and PGH(2) which are further converted to a series of prostaglandins and thromboxane A(2). Here, we report that GSH promotes concomitant formation of the current oxidative stress biomarkers malondialdehyde (MDA) and 15(S)-8-iso-prostaglandin F(2α) from AA via PGHS. This illustrates an uncommon interplay of enzymatic and chemical reactions to produce species that are considered to be exclusively produced by free-radical-catalysed reactions. We propose mechanisms for the PGHS/AA/GSH-dependent formation of MDA, 15(S)-8-iso-prostaglandin F(2α) and other F(2)-isoprostanes. These mechanisms are supported by clinical observations.

Stable isotope gas chromatography-tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD; systematic name: 1,2-3,4-5,6-7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one) is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, and cells in culture and vegetables and to possess potent antioxidative properties. Here, we describe a stable isotope gas chromatography-tandem mass spectrometry (GC-MS/MS) method for specific and sensitive determination of AECK-DD in biological samples. (13)C(2)-labeled AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected reaction monitoring of the mass transitions m/z 328 to 268 for AECK-DD and m/z 330 to 270 for [(13)C(2)]AECK-DD in the electron capture negative ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above approximately 4nM but was present in urine samples of healthy humans at a maximal concentration of 46nM. AECK-DD was detectable in rat brain at very low levels of approximately 8pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (∼1nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (∼6.8pmol/g fresh tissue).

Stable-isotope dilution GC-MS method for ethanol in vapour ethanol and microdialysis systems based on carbonate-catalyzed extractive pentafluorobenzoylation.

Common ethanol detection methods are not applicable to cell culture media and microdialysates due to interference with medium constituents including amino acids and pH indicators. We present a novel GC-MS method for the accurate and precise analysis of ethanol in cell cultures and microdialysates. The method is based on the carbonate-catalyzed extractive pentafluorobenzoylation of ethanol and deuterium-labelled ethanol serving as the internal standard and on their GC-MS analysis in the electron-capture negative-ion chemical ionization mode. The method was used to optimize experimental conditions in a custom-made ethanol vapour system utilized for studies examining ethanol influences on neuronal cell lines and in microdialysis.

Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite.

Nitrite (ONO(-)) exerts nitric oxide (NO)-related biological actions and its concentration in the circulation may be of particular importance. Nitrite is excreted in the urine. Hence, the kidney may play an important role in nitrite/NO homeostasis in the vasculature. We investigated a possible involvement of renal carbonic anhydrases (CAs) in endogenous nitrite reabsorption in the proximal tubule. The potent CA inhibitor acetazolamide was administered orally to six healthy volunteers (5 mg/kg) and nitrite was measured in spot urine samples before and after administration. Acetazolamide increased abruptly nitrite excretion in the urine, strongly suggesting that renal CAs are involved in nitrite reabsorption in healthy humans. Additional in vitro experiments support our hypothesis that nitrite reacts with CO(2), analogous to the reaction of peroxynitrite (ONOO(-)) with CO(2), to form acid-labile nitrito carbonate [ONOC(O)O(-)]. We assume that this reaction is catalyzed by CAs and that nitrito carbonate represents the nitrite form that is actively transported into the kidney. The significance of nitrite reabsorption in the kidney and the underlying mechanisms, notably a direct involvement of CAs in the reaction between nitrite and CO(2), remain to be elucidated.

Measurement of nitrite in urine by gas chromatography-mass spectrometry.

Nitric oxide (NO) is enzymatically produced from L-arginine and has a variety of biological functions. Autoxidation of NO in aqueous media yields nitrite (O = N-O(-)). NO and nitrite are oxidized in erythrocytes by oxyhemoglobin to nitrate (NO(3)(-)). Nitrate reductases from bacteria reduce nitrate to nitrite. Nitrite and nitrate are ubiquitous in nature, they are present throughout the body and they are excreted in the urine. Nitrite in urine has been used for several decades as an indicator and measure of bacteriuria. Since the identification of nitrite as a metabolite of NO, circulating nitrite is also used as an indicator of NO synthesis and is considered an NO storage form. In contrast to plasma nitrite, the significance of nitrite in the urine beyond bacteriuria is poorly investigated and understood. This chapter describes a gas chromatography-mass spectrometry (GC-MS) protocol for the quantitative determination of nitrite in urine of humans. Although the method is useful for detection and quantification of bacteriuria, the procedures described herein are optimum for urinary nitrite in conditions other than urinary tract infection. The method uses [(15)N]nitrite as internal standard and pentafluorobenzyl bromide as the derivatization agent. Derivatization is -performed on 100-µL aliquots and quantification of toluene extracts by selected-ion monitoring of m/z 46 for urinary nitrite and m/z 47 for the internal standard in the electron-capture negative-ion chemical ionization mode.

Measurement of 3-nitro-tyrosine in human plasma and urine by gas chromatography-tandem mass spectrometry.

Reaction of reactive nitrogen species (RNS), such as peroxynitrite and nitryl chloride with soluble tyrosine and tyrosine residues in proteins produces soluble 3-nitro-tyrosine and 3-nitro-tyrosino-proteins, respectively. Regular proteolysis of 3-nitro-tyrosino-proteins yields soluble 3-nitro-tyrosine. 3-Nitro-tyrosine circulates in plasma and is excreted in the urine. Both circulating and excretory 3-nitro-tyrosine are considered suitable biomarkers of nitrative stress. Tandem mass spectrometry coupled with gas chromatography (GC-MS/MS) or liquid chromatography (LC-MS/MS) is one of the most reliable analytical techniques to determine 3-nitro-tyrosine. Here, we describe protocols for the quantitative determination of soluble 3-nitro-tyrosine in human plasma and urine by GC-MS/MS.

Simultaneous UPLC-MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction.

Analysis of the endocannabinoid (EC) system's key molecules 2-arachidonoyl glycerol (2AG) and arachidonoyl ethanolamide (anandamide, AEA) is challenging due to several peculiarities. 2AG isomerizes spontaneously to its biologically inactive analogue 1-arachidonoyl glycerol (1AG) by acyl migration and it is only chromatographically distinguishable from 1AG. Matrix-effects caused primarily by co-extracted phospholipids may further compromise analysis. In addition, 2AG and 1AG are unstable under certain conditions like solvent evaporation or reconstitution of dried extracts. We examined effects of different organic solvents and their mixtures, such as toluene, ethyl acetate, and chloroform-methanol, on 2AG/1AG isomerisation, 2AG/1AG stability, and matrix-effects in the UPLC-MS/MS analysis of 2AG and AEA in human plasma. Toluene prevented, both, 2AG isomerisation to 1AG and degradation of 2AG/1AG during evaporation. Toluene extracts contain only 2% of matrix-effect-causing plasma phospholipids compared to extracts from the traditionally used solvent mixture chloroform-methanol. Toluene and all other tested organic solvents provide comparable 2AG and AEA extraction yields (60-80%). Based on these favourable toluene properties, we developed and validated a UPLC-MS/MS method with positive electrospray ionization (ESI+) that allows for simultaneous accurate and precise measurement of 2AG and AEA in human plasma. The UPLC-MS/MS method was cross-validated with a previously described fully-validated GC-MS/MS method for AEA in human plasma. A close correlation (r(2)=0.821) was observed between the results obtained from UPLC-MS/MS (y) and GC-MS/MS (x) methods (y=0.01+0.85x). The UPLC-MS/MS method is suitable for routine measurement of 2AG and AEA in human plasma samples (1 mL) in clinical settings as shown by quality control plasma samples processed over a period of 100 days. The UPLC-MS/MS method was further extended to human urine. In urine, AEA was not detectable and 2AG was detected in only 3 out of 19 samples from healthy subjects at 160, 180 and 212 pM corresponding to 12.3, 14.5 and 9.9 pmol/mmol creatinine, respectively.

¹8O-Labeled nitrous acid and nitrite: Synthesis, characterization, and oxyhemoglobin-catalyzed oxidation to ¹8O-labeled nitrate.

We describe a simple laboratory method for specific labeling of nitrite with ¹8O for use in chemical and biochemical studies in the area of nitric oxide research. NaNO2 (0.1 mmol) is diluted in H2¹8O (45 µl) and acidified with HCl (1 µl, 5 M), and the solution is allowed to equilibrate. Subsequently, the sample is mixed by vortexing with ethyl acetate (500 µl), and the organic phase is dried over anhydrous Na2SO(4). Ethyl acetate is evaporated to dryness, and the residue is reconstituted in phosphate-buffered saline. In human blood hemolysate, oxyhemoglobin (HbFe¹6O2) was shown to oxidize N¹8O2⁻ to ¹6ON¹8O2⁻.

Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: a comprehensive review from an analytical and biological perspective.

The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are physiologically occurring, biologically active compounds on CB(1) and CB(2) receptors with multiple physiological functions. AEA and 2AG have been identified and quantified in many mammalian biological fluids and tissues, such as human plasma, adipocytes, tissues and tissue microdialysates, at concentrations in the picomolar-to-nanomolar range under basal conditions. In this article, recently published chromatographic and mass spectrometric analytical methods, i.e., HPLC with fluorescence or ultraviolet detection, LC-MS, LC-MS/MS, GC-MS and GC-MS/MS, are reviewed and discussed, notably from the quantitative point of view. We focus on and emphasize the particular importance of blood sampling, sample storage and work-up including solvent and solid-phase extraction and derivatization procedures, matrix-effects, and stability of analytes. As 2AG spontaneously isomerizes to its CB(1)/CB(2) receptors biologically inactive 1-arachidonoyl glycerol (1AG) by acyl migration, this phenomenon and its particular importance for accurate quantification of 2AG are discussed in detail. Due to the electrical neutrality of AEA and 2AG their solvent extraction by toluene offers the least matrix-effect and minimum isomerization. LC-MS/MS is the most frequently used analytical technique for AEA and 2AG. At present, the utility of the GC-MS/MS methodology seems to be limited to AEA measurement in human plasma, bronchoalveolar liquid (BAL) and microdialysate samples. Despite great instrumental advances in the LC-MS/MS methodology, sampling and sample treatment remains one of the most crucial analytical steps in 2AG analysis. Extension of the LC-MS/MS methodology, for instance to microdialysate and BAL samples from clinical studies, is a big analytical challenge in endocannabinoid analysis in clinical settings. Currently available LC-MS/MS and GC-MS/MS methods should be useful to investigate the metabolism of AEA and 2AG beyond hydrolysis, i.e., by ß- and ω-oxidation pathways.

Quantification of acetaminophen (paracetamol) in human plasma and urine by stable isotope-dilution GC-MS and GC-MS/MS as pentafluorobenzyl ether derivative.

We report on the quantitative determination of acetaminophen (paracetamol; NAPAP-d(0)) in human plasma and urine by GC-MS and GC-MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode after derivatization with pentafluorobenzyl (PFB) bromide (PFB-Br). Commercially available tetradeuterated acetaminophen (NAPAP-d(4)) was used as the internal standard. NAPAP-d(0) and NAPAP-d(4) were extracted from 100-µL aliquots of plasma and urine with 300 µL ethyl acetate (EA) by vortexing (60s). After centrifugation the EA phase was collected, the solvent was removed under a stream of nitrogen gas, and the residue was reconstituted in acetonitrile (MeCN, 100 µL). PFB-Br (10 µL, 30 vol% in MeCN) and N,N-diisopropylethylamine (10 µL) were added and the mixture was incubated for 60 min at 30 °C. Then, solvents and reagents were removed under nitrogen and the residue was taken up with 1000 µL of toluene, from which 1-µL aliquots were injected in the splitless mode. GC-MS quantification was performed by selected-ion monitoring ions due to [M-PFB](-) and [M-PFB-H](-), m/z 150 and m/z 149 for NAPAP-d(0) and m/z 154 and m/z 153 for NAPAP-d(4), respectively. GC-MS/MS quantification was performed by selected-reaction monitoring the transition m/z 150 → m/z 107 and m/z 149 → m/z 134 for NAPAP-d(0) and m/z 154 → m/z 111 and m/z 153 → m/z 138 for NAPAP-d(4). The method was validated for human plasma (range, 0-130 µM NAPAP-d(0)) and urine (range, 0-1300 µM NAPAP-d(0)). Accuracy (recovery, %) ranged between 89 and 119%, and imprecision (RSD, %) was below 19% in these matrices and ranges. A close correlation (r>0.999) was found between the concentrations measured by GC-MS and GC-MS/MS. By this method, acetaminophen can be reliably quantified in small plasma and urine sample volumes (e.g., 10 µL). The analytical performance of the method makes it especially useful in pediatrics.

In-source formation of N-acetyl-p-benzoquinone imine (NAPQI), the putatively toxic acetaminophen (paracetamol) metabolite, after derivatization with pentafluorobenzyl bromide and GC-ECNICI-MS analysis.

Pentafluorobenzyl (PFB) bromide (PFB-Br) is a versatile derivatization reagent for numerous classes of compounds. Under electron-capture negative-ion chemical ionization (ECNICI) conditions PFB derivatives of acidic compounds readily and abundantly ionize to produce intense anions due to [M-PFB](-). In the present article we investigated the PFB-Br derivatization of unlabelled acetaminophen (N-acetyl-p-aminophenol, NAPAP-d(0); paracetamol; MW 151) and tetradeuterated acetaminophen (NAPAP-d(4); MW 155) in anhydrous acetonitrile and their GC-ECNICI-MS behavior using methane as the buffer gas. In addition to the expected anions [M-PFB](-) at m/z 150 from NAPAP-d(0) and m/z 154 from NAPAP-d(4), we observed highly reproducibly almost equally intense anions at m/z 149 and m/z 153, respectively. Selected ion monitoring of these ions is suitable for specific and sensitive quantification of acetaminophen in human plasma and urine. Detailed investigations suggest in-source formation of N-acetyl-p-benzoquinone imine (NAPQI; MW 149), the putatively toxic acetaminophen metabolite, from the PFB ether derivative of NAPAP. GC-ECNICI-MS of non-derivatized NAPAP did not produce NAPQI. The peak area ratio of m/z 149 to m/z 150 and of m/z 153 to m/z 154 decreased with increasing ion-source temperature in the range 100-250°C. Most likely, NAPQI formed in the ion-source captures secondary electrons to become negatively charged (i.e., [NAPQI](-)) and thus detectable. Formation of NAPQI was not observed under electron ionization (EI) conditions, i.e., by GC-EI-MS, from derivatized and non-derivatized NAPAP. NAPQI was not detectable in flow injection analysis LC-MS of native NAPAP in positive electrospray ionization (ESI) mode, whereas in negative ESI mode low extent NAPQI formation was observed (<5%). Our results suggest that oxidation of drug derivatives in the ion-sources of mass spectrometers may form intermediates that are produced from activated drugs in enzyme-catalyzed reactions.

Simultaneous gas chromatography-tandem mass spectrometry quantification of symmetric and asymmetric dimethylarginine in human urine.

Previously, we demonstrated the utility of a gas chromatography-tandem mass spectrometry (GC-MS/MS) method for the quantitative determination of asymmetric dimethylarginine (ADMA) in biological samples. Here we report the extension of this method to symmetric dimethylarginine (SDMA) in human urine. SDMA and ADMA were simultaneously quantitated in urine by using their in situ prepared trideuteromethyl esters as internal standards. The GC-MS/MS method was validated for SDMA and ADMA in spot urine samples of 19 healthy adults. In these samples, the creatinine-corrected excretion rate was 3.23±0.63 µmol/mmol for SDMA and 3.14±0.98 µmol/mmol for ADMA.

Stable-isotope dilution GC-MS approach for nitrite quantification in human whole blood, erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: nitrite distribution in human blood.

Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC-MS using their (15)N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC-MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0-4°C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50°C). Potassium ferricyanide (K(3)Fe(CN)(6)) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC-MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486±280 nM in whole blood, 672±496 nM in plasma (C(P)), and 620±350 nM in erythrocytes (C(E)). The C(E)-to-C(P) ratio was 0.993±0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC-MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood compartments under basal conditions and during hyperemia.