Development, validation and biomedical applications of stable-isotope dilution GC-MS and GC-MS/MS techniques for circulating malondialdehyde (MDA) after pentafluorobenzyl bromide derivatization: MDA as a biomarker of oxidative stress and its relation to 15(S)-8-iso-prostaglandin F2α and nitric oxide (NO).

Malondialdehyde (MDA, CH2(CHO)2) is one of the best investigated and most frequently measured biomarkers of lipid peroxidation in biological fluids, a constituent of the so called thiobarbituric acid reactive substances (TBARS). The reaction of thiobarbituric acid with MDA and other carbonyl compounds is the basis for the batch TBARS assay, one of the most commonly and widely used assays of oxidative stress. Yet, the TBARS assay lacks specificity even if combined with HPLC separation prior to visible absorbance or fluorescence detection. In this article, we report highly specific and sensitive stable-isotope dilution GC-MS and GC-MS/MS methods for the quantitative determination of MDA in human plasma (0.1 mL). These methods utilize the acidity (pKa, 4.46) of the two methylene H protons of MDA in aqueous solution, which are as acidic as acetic acid. Endogenous MDA in native plasma and the externally added internal standard [1,3-(2)H2]-MDA (d2-MDA, CH2(CDO)2) are derivatized in aqueous acetone (400 µL) with pentafluorobenzyl (PFB) bromide (10 µL). The reaction products were identified as C(PFB)2(CHO)2 (molecular weight, 432) and C(PFB)2(CDO)2) (molecular weight, 434), respectively. After solvent extraction with toluene (1 mL) quantification is performed by selected-ion monitoring (SIM) in GC-MS and by selected-reaction monitoring (SRM) in GC-MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode. In the SIM mode, the anions [M-PFB](-) at m/z 251 for MDA and m/z 253 for d2-MDA are detected. In the SRM mode, the mass transitions m/z 251 to m/z 175 for MDA and m/z 253 to m/z 177 for d2-MDA are monitored. The method was thoroughly validated in human plasma. Potential interfering substances including anticoagulants and commercially available monovettes commonly used for blood sampling were tested. The lowest MDA concentrations were measured in serum followed by heparinized and EDTA plasma. The GC-MS and GC-MS/MS methods were found to be specific, precise, accurate and sensitive. Thus, the LOD of the GC-MS/MS method was determined to be 2 amol (2 × 10(-18)mol) MDA. The GC-MS/MS method is exceedingly useful in clinical settings. We report several biomedical applications and discuss the utility of circulating MDA as a biomarker of lipid peroxidation, especially in long-term clinical studies, and its relation to the F2-isoprostane 15(S)-8-iso-prostaglandin F2α and nitric oxide (NO).

Trapping of NAPQI, the intermediate toxic paracetamol metabolite, by aqueous sulfide (S²â») and analysis by GC-MS/MS.

NAPQI, i.e., N-acetyl-p-benzoquinone imine, is considered the toxic metabolite of the widely used analgesic drug paracetamol (acetaminophen, APAP). Due to its high reactivity towards nucleophiles both in low- and high-molecular-mass biomolecules, NAPQI is hardly detectable in its native form. Upon conjugation with glutathione, NAPQI is finally excreted in the urine as the paracetamol mercapturic acid. Thus, determination of paracetamol mercapturate may provide a measure of in vivo NAPQI formation. In this work, we propose the use of Na2S in aqueous solution to trap NAPQI and to analyze the reaction product, i.e., 3-thio-paracetamol, together with paracetamol by GC-MS/MS in the electron-capture negative-ion chemical ionization mode after solvent extraction with ethyl acetate and derivatization with pentafluorobenzyl bromide. In mechanistic studies, we used newly synthesized N-acetyl-p-[2,3,5,6-(2)H4]benzoquinone imine (d4-NAPQI). In quantitative analyses, N-(4-hydroxyphenyl)-[2,3,5,6-(2)H4]acetamide (d4-APAP) was used as the internal standard both for NAPQI and APAP. 3-Thio-d3-paracetamol, prepared from d4-NAPQI and Na2S, may also be useful as an internal standard. We showed NAPQI in vitro formation from APAP by recombinant cyclooxygenase-1 as well as by dog liver homogenate. In vivo formation of NAPQI was demonstrated in mice given paracetamol intraperitoneally (about 150 mg/kg).

Effects of paracetamol on NOS, COX, and CYP activity and on oxidative stress in healthy male subjects, rat hepatocytes, and recombinant NOS.

Paracetamol (acetaminophen) is a widely used analgesic drug. It interacts with various enzyme families including cytochrome P450 (CYP), cyclooxygenase (COX), and nitric oxide synthase (NOS), and this interplay may produce reactive oxygen species (ROS). We investigated the effects of paracetamol on prostacyclin, thromboxane, nitric oxide (NO), and oxidative stress in four male subjects who received a single 3 g oral dose of paracetamol. Thromboxane and prostacyclin synthesis was assessed by measuring their major urinary metabolites 2,3-dinor-thromboxane B2 and 2,3-dinor-6-ketoprostaglandin F(1α), respectively. Endothelial NO synthesis was assessed by measuring nitrite in plasma. Urinary 15(S)-8-iso-prostaglanding F(2α) was measured to assess oxidative stress. Plasma oleic acid oxide (cis-EpOA) was measured as a marker of cytochrome P450 activity. Upon paracetamol administration, prostacyclin synthesis was strongly inhibited, while NO synthesis increased and thromboxane synthesis remained almost unchanged. Paracetamol may shift the COX-dependent vasodilatation/vasoconstriction balance at the cost of vasodilatation. This effect may be antagonized by increasing endothelial NO synthesis. High-dosed paracetamol did not increase oxidative stress. At pharmacologically relevant concentrations, paracetamol did not affect NO synthesis/bioavailability by recombinant human endothelial NOS or inducible NOS in rat hepatocytes. We conclude that paracetamol does not increase oxidative stress in humans.

LC-MS/MS and GC-MS/MS measurement of plasma and urine di-paracetamol and 3-nitro-paracetamol: proof-of-concept studies on a novel human model of oxidative stress based on oral paracetamol administration.

Paracetamol (acetaminophen) is a widely used safe analgesic drug when administered at therapeutic doses. Given the chemical reactivity of its phenolic group towards electrophilic species, we assumed that detection of paracetamol metabolites distinctly different from its known phase I metabolite N-acetyl-p-benzoquinone imine (NAPQI) and the phase II glucuronic, sulfuric and mercapturic acids in biological samples upon oral administration of paracetamol (e.g., a 500-mg tablet) may represent a novel model of oxidative stress in humans. Such potential paracetamol metabolites are di-paracetamol and 3-nitro-paracetamol, in analogy to the well-investigated endogenous biomarkers di-tyrosine and 3-nitro-tyrosine. Di-paracetamol and 3-nitro-paracetamol are known to be formed both by enzymatic and non-enzymatic routes. In the present work we report on mouse and human pilot studies on the formation and appearance of di-paracetamol and 3-nitro-paracetamol in blood of mice intraperitoneally administered paracetamol, as well as in plasma and urine samples of healthy subjects who received a 500-mg paracetamol tablet or placebo. For the analysis of di-paracetamol and 3-nitro-paracetamol in plasma and urine samples, analytes were extracted by solvent extraction with ethyl acetate and subsequently analyzed by LC-MS/MS without and with derivatization with pentafluorobenzyl bromide. GC-MS/MS was used to detect 3-nitro-paracetamol and quantify paracetamol as pentafluorobenzyl derivatives. Our studies indicate that di-paracetamol and 3-nitro-paracetamol appear in plasma and urine when paracetamol is given orally to healthy humans at the therapeutic dosage of 5-7 mg/kg. The molar ratio of di-paracetamol to paracetamol in urine was determined to be 1:535 in the paracetamol group and 1:6844 in the placebo group; the molar ratio of 3-nitro-paracetamol to paracetamol in urine was determined to be 1:199 in the paracetamol group and 1:8657 in the placebo group. Our studies suggest that a fraction of circulating and excretory di-paracetamol and 3-nitro-paracetamol may be formed artefactually during sample workup including derivatization. Further studies based on the quantitative determination of di-paracetamol and 3-nitro-paracetamol in biological samples by LC-MS/MS and/or GC-MS/MS using stable-isotope labeled analogues as internal standards are warranted to test the utility of paracetamol as a probe of oxidative stress in animals and in humans in health and disease.

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.

LC-MS/MS analysis of uncommon paracetamol metabolites derived through in vitro polymerization and nitration reactions in liquid nitrogen.

Paracetamol (acetaminophen, APAP) is a commonly used analgesic drug. Known paracetamol metabolites include the glucuronide, sulfate and mercapturate. N-Acetyl-benzoquinonimine (NAPQI) is considered the toxic intermediate metabolite of paracetamol. In vitro and in vivo studies indicate that paracetamol is also metabolized to additional poorly characterized metabolites. For example, metabolomic studies in urine samples of APAP-treated mice revealed metabolites such as APAP-sulfate-APAP and APAP-S-S-APAP in addition to the classical phase II metabolites. Here, we report on the development and application of LC-MS and LC-MS/MS approaches to study reactions of unlabelled and (2)H-labelled APAP with unlabelled and (15)N-labelled nitrite in aqueous phosphate buffers (pH 7.4) upon their immersion into liquid nitrogen (-196°C). In mechanistic studies, these reactions were also studied in aqueous buffer prepared in (18)O-labelled water. LC-MS and LC-MS/MS analyses were performed on a reverse-phase material (C18) using gradient elution (2mM ammonium acetate/acetonitrile), in positive and negative electrospray mode. We identified a series of APAP metabolites including di-, tri- and tetra-APAP, mono- and di-nitro-APAP and nitric ester of di-APAP. Our study indicates that nitrite induces oxidation, i.e., polymerization and nitration of APAP, when buffered APAP/nitrite solutions are immersed into liquid nitrogen. These reactions are specific for nitrite with respect to nitrate and do not proceed via intermediate formation of NAPQI. Potassium ions and physiological saline but not thiols inhibit nitrite- and shock-freeze-induced reactions of paracetamol. The underlying mechanism likely involves in situ formation of NO2 radicals from nitrite secondary to profound pH reduction (down to pH 1) and disproportionation. Polymeric paracetamol species can be analyzed as pentafluorobenzyl derivatives by LC-MS but not by GC-MS.

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.