Pentafluorobenzyl Research Articles

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 (60 s). 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.

Analysis of epoxyeicosatrienoic acids by chiral liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry using [13C]-analog internal standards.

The metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) is thought to be mediated primarily by the cytochromes P450 (P450s) from the 2 family (2C9, 2C19, 2D6, and 2J2). In contrast, P450s of the 4 family are primarily involved in omega oxidation of AA (4A11 and 4A22). The ability to determine enantioselective formation of the regioisomeric EETs is important in order to establish their potential biological activities and to asses which P450 isoforms are involved in their formation. It has been extremely difficult to analyze individual EET enantiomers in biological fluids because they are present in only trace amounts and they are extremely difficult to separate from each other. In addition, the deuterium-labeled internal standards that are commonly used for stable isotope dilution liquid chromatography/mass spectrometry (LC/MS) analyses have different LC retention times when compared with the corresponding protium forms. Therefore, quantification by LC/MS-based methodology can be compromised by differential suppression of ionization of the closely eluting isomers. We report the preparation of [(13)C(20)]-EET analog internal standards and the use of a validated high-sensitivity chiral LC/electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the trace analysis of endogenous EETs as their pentafluorobenzyl (PFB) ester derivatives. The assay was then used to show the exquisite enantioselectivity of P4502C19-, P4502D6-, P4501A1-, and P4501B1-mediated conversion of AA into EETs and to quantify the enantioselective formation of EETs produced by AA metabolism in a mouse epithelial hepatoma (Hepa) cell line.

Unique pentafluorobenzylation and collision-induced dissociation for specific and accurate GC–MS/MS quantification of the catecholamine metabolite 3,4-dihydroxyphenylglycol (DHPG) in human urine

In the human body, the catecholamine norepinephrine is mainly metabolized to 3,4-dihydroxyphenylglycol (DHPG) which therefore serves as an important biomarker for norepinephrine's metabolism. Most data on DHPG concentrations in human plasma and urine has been generated by using HPLC-ECD or GC–MS technologies. Here, we describe a stable-isotope dilution GC–MS/MS method for the quantitative determination of DHPG in human urine using trideutero-DHPG (d 3-DHPG) as internal standard and a two-step derivatization process with pentafluorobenzyl bromide (PFB-Br) and N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Two pentafluorobenzyl (PFB) trimethylsilyl (TMS) derivatives were obtained and identified, i.e., two isomeric DHPG-PFB-(TMS) 3 derivatives and the later eluting DHPG-tetrafluorobenzyl-(TMS) 2 derivative, i.e., DHPG-TFB-(TMS) 2. To our knowledge the DHPG-TFB-(TMS) 2 derivative and the underlying reaction have not been reported previously. In this reaction both vicinal aromatic hydroxyl groups of DHPG react with PFB-Br to form a heterocyclic seven-membered [1,4]dioxepin compound. The DHPG-TFB-(TMS) 2 derivative was used for quantitative GC–MS/MS analysis in the electron-capturing negative-ion chemical ionization mode by selected-reaction monitoring of m/ z 351 from m/ z 401 for DHPG and of m/ z 352 from m/ z 404 for d 3-DHPG. Validation experiments on human urine samples spiked with DHPG in a narrow (0–33 nM) and a wide range (0–901 nM) revealed high recovery (86–104%) and low imprecision (RSD; 0.01–2.8%). LOD and relative LLOQ (rLLOQ) values of the method for DHPG were determined to be 76 amol and 9.4%, respectively. In urine of 28 patients suffering from chronic inflammatory rheumatic diseases, DHPG was measured at a mean concentration of 238 nM (38.3 μg/g creatinine). The DHPG concentration in the respective control group of 40 healthy subjects was measured to be 328 nM (39.2 μg/g creatinine). Given the unique derivatization reaction and collision-induced dissociation, and the straightforwardness the present method is highly specific, accurate, precise, and should be useful in clinical settings.

Quantification of Carbonate by Gas Chromatography−Mass Spectrometry

Carbon dioxide and carbonates are widely distributed in nature, are constituents of inorganic and organic matter, and are essential in vegetable and animal organisms. CO(2) is the principal greenhouse gas in the atmosphere. In human blood, CO(2)/HCO(3)(-) is an important buffering system. Quantification of bicarbonate and carbonate in inorganic and organic matter and in biological fluids such as blood or blood plasma by means of the GC-MS technology has been impossible so far, presumably because of the lack of suitable derivatization reactions to produce volatile and thermally stable derivatives. Here, a novel derivatization reaction is described for carbonate that allows for its quantification in aqueous alkaline solutions and alkalinized plasma and urine. Carbonate in acetonic solutions of these matrices (1:4 v/v) and added (13)C-labeled carbonate for use as the internal standard were heated in the presence of the derivatization agent pentafluorobenzyl (PFB) bromide for 60 min and 50 °C. Investigations with (12)CO(3)(2-), (13)CO(3)(2-), (CH(3))(2)CO, and (CD(3))(2)CO in alkaline solutions and GC-MS and GC-MS/MS analyses under negative-ion chemical ionization (NICI) or electron ionization (EI) conditions of toluene extracts of the reactants revealed formation of two minor [i.e., PFB-OCOOH and O=CO(2)-(PFB)(2)] and two major [i.e., CH(3)COCH(2)-C(OH)(OPFB)(2) and CH(3)COCH=C(OPFB)(2)] carbonate derivatives. The latter have different retention times (7.9 and 7.5 min, respectively) but virtually identical EI and NICI mass spectra. It is assumed that CH(3)COCH(2)-C(OH)(OPFB)(2) is formed from the reaction of the carbonate dianion with two molecules of PFB bromide to form the diPFB ester of carbonic acid, which further reacts with one molecule of acetone. Subsequent loss of water finally generates the major derivative CH(3)COCH=C(OPFB)(2). This derivatization reaction was utilized to quantify total CO(2)/HCO(3)(-)/CO(3)(2-) (tCO(2)) in human plasma and urine by GC-MS in the NICI mode by selected ion monitoring of the anions [M-H](-) of CH(3)COCH=C(OPFB)(2) at m/z 461 for the endogenous species and m/z 462 for the internal standard (13)CO(3)(2-). Oral intake of the carboanhydrase inhibitor drug acetazolamide by two healthy volunteers resulted in temporary increased excretion of tCO(2) in the urine. The method is specific for carbonate, accurate, sensitive and should be applicable to various matrices including human fluids and environmental samples.

GC–MS determination of creatinine in human biological fluids as pentafluorobenzyl derivative in clinical studies and biomonitoring: Inter-laboratory comparison in urine with Jaffé, HPLC and enzymatic assays

In consideration of its relatively constant urinary excretion rate, creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous biomolecules. Assays based on the reaction of creatinine and picric acid first reported by Jaffé in 1886 still belong to the most frequently used laboratory approaches for creatinine measurement in urine. Further analytical methods for creatinine include HPLC–UV, GC–MS, and LC–MS and LC–MS/MS approaches. In the present article we report on the development, validation and biomedical application of a new GC–MS method for the reliable quantitative determination of creatinine in human urine, plasma and serum. This method is based on the derivatization of creatinine (d 0-Crea) and the internal standard [ methyl-trideutero]creatinine (d 3-Crea) with pentafluorobenzyl (PFB) bromide in the biological sample directly or after dilution with phosphate buffered saline, extraction of the reaction products with toluene and quantification in 1-μl aliquots of the toluene extract by selected-ion monitoring of m/ z 112 for d 0-Crea-PFB and m/ z 115 for d 3-Crea-PFB in the electron-capture negative-ion chemical ionization mode. The limit of detection of the method is 100 amol of creatinine. In an inter-laboratory study on urine samples from 100 healthy subjects, the GC–MS method was used to test the reliability of currently used Jaffé, enzymatic and HPLC assays in clinical and occupational studies. The results of the inter-laboratory study indicate that all three tested methods allow for satisfactory quantification of creatinine in human urine. The GC–MS method is suitable for use as a reference method for urinary creatinine in humans. In serum, creatine was found to contribute to creatinine up to 20% when measured by the present GC–MS method. The application of the GC–MS method can be extended to other biological samples such as saliva.

Liquid chromatography–mass spectrometry (LC–MS) of steroid hormone metabolites and its applications

Advances in liquid chromatography–mass spectrometry (LC–MS) can be used to measure steroid hormone metabolites in vitro and in vivo. We find that LC–electrospray ionization (ESI)-MS using a LCQ ion trap mass spectrometer in the negative ion mode can be used to monitor the product profile that results from 5α-dihydrotestosterone (DHT)-17β-glucuronide, DHT-17β-sulfate, and tibolone-17β-sulfate reduction catalyzed by human members of the aldo–keto reductase (AKR) 1C subfamily and assign kinetic constants to these reactions. We also developed a stable isotope dilution LC–electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the quantitative analysis of estrone (E1) and its metabolites as pentafluorobenzyl (PFB) derivatives in human plasma in the attomole range. The limit of detection for E1-PFB was 740attomole on column. Separations can be performed using normal-phase LC because ionization takes place in the gas phase rather than in solution. This permits efficient separation of the regioisomeric 2- and 4-methoxy-E1. The method was validated for the simultaneous analysis of plasma E2 and its metabolites: 2-methoxy-E2, 4-methoxy-E2, 16α-hydroxy-E2, estrone (E1), 2-methoxy-E1, 4-methoxy-EI, and 16α-hydroxy-E1 from 5pg/mL to 2000pg/mL. Our LC–MS methods have sufficient sensitivity to detect steroid hormone levels in prostate and breast tumors and should aid their molecular diagnosis and treatment.

9016 Nutritional assessment on hospitalised lung cancer patients

A high-performance liquid chromatography (HPLC) and gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI-MS) method was developed for the analysis of the combined antitumor drug S-1 (tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate[ and active metabolite 5-fluorouracil in human plasma and urine. Tegafur was fractionated from biological fluids by extraction with dichloromethane and analyzed by HPLC. 5-Fluorouracil and 5-chloro-2,4-dihydroxypyridine were extracted with ethyl acetate from the residual layer after extraction of tegafur, and converted to pentafluorobenzyl (PFB) derivatives. Potassium oxonate was cleaned up with an anion-exchange column (Bond Elut NH2). The extracted potassium oxonate was degraded to 5-azauracil and converted to PFB derivatives. The PFB derivatives were analyzed by GC-NICI-MS. A stable isotope was employed as the internal standard in the GC-NICI-MS analysis. The limits of quantitation of tegafur, 5-fluorouracil, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in plasma were 10, 1, 2 and 1 ng/ml, respectively. The reproducibility of the analytical method according to the statistical coefficients is ∼10%. The accuracy of the method is good; that is, the relative error is <10%. The methods were applied to pharmacokinetic studies of S-1 in patients.

Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified α-linolenic acid in unanesthetized rats

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), long-chain n-3 PUFAs important for brain and heart function, can be obtained from dietary fish products or by liver synthesis from alpha-linolenic acid (alpha-LNA). Their daily human dietary requirements are not clear, and their liver synthesis rates in humans and nonhumans are unknown. We estimated whole-body (presumably liver) synthesis rates in unanesthetized rats by infusing [U-(13)C]alpha-LNA intravenously for 2 h and measuring labeled and unlabeled n-3 PUFA in arterial plasma using negative chemical ionization GC-MS. Newly synthesized esterified [(13)C]DHA, [(13)C]EPA, and [(13)C]docosapentaenoic acid (DPA) appeared in arterial plasma after 60 min of infusion, then their concentrations rose in an S-shaped manner. Esterified concentration x plasma volume data were fit with a sigmoidal equation, whose peak first derivatives provided synthesis rates of unlabeled EPA, DPA, and DHA equal to 8.40, 6.27, and 9.84 mumol/day, respectively. The DHA synthesis rate exceeded the published daily rat brain DHA consumption rate by 30-fold, suggesting that liver synthesis from alpha-LNA could maintain brain DHA homeostasis were DHA absent from the diet. This stable isotope infusion method could be used to quantify whole-body DHA synthesis rates in human subjects.

Specific GC–MS/MS stable-isotope dilution methodology for free 9- and 10-nitro-oleic acid in human plasma challenges previous LC–MS/MS reports

Nitrated unsaturated fatty acids including nitro-oleic acid (NO 2-OA) have been measured in human blood samples in their free and esterified forms. Plasma concentrations in healthy subjects have been reported to be of the order of 600 nM for free NO 2-OA and 300 nM for esterified NO 2-OA, as measured by LC–MS/MS. In the present article we report a GC–MS/MS method for the specific and accurate quantification of two NO 2-OA isomers, i.e., 9-NO 2-OA and 10-NO 2-OA, in human plasma using newly prepared, isolated, characterized and standardized 15N-labeled analogs. This method involves SPE extraction of fatty acids from slightly acidified plasma samples (pH 5), conversion to their pentafluorobenzyl (PFB) esters, isolation by HPLC, solvent extraction from a single HPLC fraction and GC–MS/MS analysis in the electron capture negative-ion chemical ionization (ECNICI) mode. Quantification was performed by selected-reaction monitoring (SRM) of m/ z 46 ([NO 2] −) and m/ z 47 ([ 15NO 2] −) produced by collision-induced dissociation (CID) from the parent ions [M−PFB] − at m/ z 326 for endogenous 9-NO 2-OA and 10-NO 2-OA and m/ z 327 for the internal standards 9- 15NO 2-OA and 10- 15NO 2-OA. We partially validated the GC–MS/MS method for 9-NO 2-OA and 10-NO 2-OA in human plasma and quantified these nitro-oleic species in plasma of 15 healthy volunteers. We identified two isomers, i.e., 9-NO 2-OA and 10-NO 2-OA, in human plasma under physiological conditions and found these nitrated fatty acids at a mean concentration of 1 nM each. This concentration is about 600 times lower than that reported by others using LC–MS/MS. Our GC–MS/MS studies on nitro-oleic acid and 3-nitrotyrosine suggest that the extent of nitration of biomolecules such as unsaturated fatty acids and tyrosine is very low in health. In this article we discuss analytical and biological ramifications potentially associated with measurement of nitrated biomolecules in biological systems.

Modeling GC‐ECD retention times of pentafluorobenzyl derivatives of phenol by using artificial neural networks

The depicted retention times (RTs) of an electron capture detection (ECD) system is predicted for a set of 37 pentafluorobenzyl (PFB) derivatives of phenol in a semi-polar column, DB-1701 (14% cyanopropylphenyl and 86% dimethyl-polysiloxane). Among a large number of descriptors, four parameters categorized as electronic, topological, geometric, and hybrid (geometric and topological) descriptors are chosen using stepwise multiple regression technique. Each molecular descriptor in this model was disputed to unfold the relationship between molecular structures and their RTs. The descriptors occurring in the multiple linear regression (MLR) model were considered as inputs for developing the back propagation artificial neural networks (BPANN). The artificial neural network (ANN) model shows superiority over the MLR by decerning 91.9% for different classes of molecules in confusion matrix. This refers to the fact that the retention behaviors of molecules display nonlinear characteristics. The accuracy of 4-4-1 BPANN model was illustrated using leave-one-out (LOO), leave-multiple-out (LMO) cross-validations, and Y-randomization. Moreover, the mean effect of descriptors betrays that descriptor Ss is the most indispensable factor affecting the retention behavior of molecules.

Ionization Mechanism of Negative Ion-Direct Analysis in Real Time: A Comparative Study with Negative Ion-Atmospheric Pressure Photoionization

The ionization mechanism of negative ion-direct analysis in real time (NI-DART) has been investigated using over 42 compounds, including fullerenes, perfluorocarbons (PFC), organic explosives, phenols, pentafluorobenzyl (PFB) derivatized phenols, anilines, and carboxylic acids, which were previously studied by negative ion-atmospheric pressure photoionization (NI-APPI). NI-DART generated ionization products similar to NI-APPI, which led to four ionization mechanisms, including electron capture (EC), dissociative EC, proton transfer, and anion attachment. These four ionization mechanisms make both NI-DART and NI-APPI capable of ionizing a wider range of compounds than negative ion-atmospheric pressure chemical ionization (APCI) or negative ion-electrospray ionization (ESI). As the operation of NI-DART is much easier than that of NI-APPI and the gas-phase ion chemistry of NI-DART is more easily manipulated than that of NI-APPI, NI-DART can be therefore used to study in detail the ionization mechanism of LC/NI-APPI-MS, which would be a powerful methodology for the quantification of low-polarity compounds. Herein, one such application has been further demonstrated in the detection and identification of background ions from LC solvents and APPI dopants, including water, acetonitrile, chloroform, methylene chloride, methanol, 2-propanol, hexanes, heptane, cyclohexane, acetone, tetrahydrofuran (THF), 1,4-dioxane, toluene, and anisole. Possible reaction pathways leading to the formation of these background ions were further inferred. One of the conclusions from these experiments is that THF and 1,4-dioxane are inappropriate to be used as solvents and/or dopants for LC/NI-APPI-MS due to their high reactivity with source basic ions, leading to many reactant ions in the background.

Whole body distribution of deuterated linoleic and α-linolenic acids and their metabolites in the rat

Little is known about the uptake or metabolism of essential fatty acids (EFAs) in various mammalian organs. Thus, the distribution of deuterated alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) and their metabolites was studied using a stable isotope tracer technique. Rats were orally administered a single dose of a mixture (20 mg each) of ethyl D5-18:3n-3 and D5-18:2n-6, and 25 tissues per animal were analyzed for D5-labeled PUFAs at 4, 8, 24, 96, 168, 240, 360, and 600 h after dosing. Plasma, stomach, and spleen contained the highest concentrations of labeled precursors at the earliest time points, whereas other internal organs and red blood cells reached their maximal concentrations at 8 h. The time-course data were consistent with liver metabolism of EFAs, but local metabolism in other tissues could not be ruled out. Brain, spinal cord, heart, testis, and eye accumulated docosahexaenoic acid with time, whereas skin accumulated mainly 20:4n-6. On average, approximately 16-18% of the D5-18:3n-3 and D5-18:2n-6 initial dosage was eventually accumulated in tissues, principally in adipose, skin, and muscle. Approximately 6.0% of D5-18:3n-3 and 2.6% of D5-18:2n-6 were elongated/desaturated and stored, mainly in muscle, adipose, and the carcass. The remaining 78% of both precursors was apparently catabolized or excreted.

Simultaneous Determination of Salsolinol Enantiomers and Dopamine in Human Plasma and Cerebrospinal Fluid by Chemical Derivatization Coupled to Chiral Liquid Chromatography/Electrospray Ionization-Tandem Mass Spectrometry

A sensitive, specific, and robust method to simultaneously determine enantiomeric salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, SAL), a potential biomarker implicated in alcohol-related neurotoxicity in a stereoselective manner, and its precursor dopamine (DA) has been developed using simple chemical derivatization and chiral separation coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS). SAL enantiomers and DA were converted to stable pentafluorobenzyl (PFB) derivatives directly from aqueous media. Bulky PFB groups introduced into the SAL structure enabled baseline separation of SAL stereoisomers on a chiral column without cumbersome chiral derivatization to unstable SAL diastereomers. Subsequent analysis by ESI-MS/MS with multiple reaction monitoring (MRM) in the presence of deuterium-labeled internal standards allowed specific detection of both derivatives with a wide dynamic range (SAL, 0.5-5000 pg; DA, 0.02-20 ng). The limit of quantitation assayed in the plasma matrix was below 10 pg for each SAL enantiomer and 100 pg for DA. Both coefficient of variance and error for inter- and intraday measurements in the blank plasma were less than 10% for SAL and DA in the concentration range of 10-4000 pg/mL and 0.1-8 ng/mL, respectively. This strategy enabled routine and specific determination of both SAL enantiomers and DA from 0.5 mL of human plasma and cerebrospinal fluid, which has not been possible using existing methodologies.

Identification of Novel Autoxidation Products of the ω-3 Fatty Acid Eicosapentaenoic Acid in Vitro and in Vivo

Increased intake of fish oil rich in the omega-3 fatty acids eicosapentaenoic acid (EPA, C20:5 omega-3) and docosahexaenoic acid (DHA, C22:6 omega-3) reduces the incidence of human disorders such as atherosclerotic cardiovascular disease. However, mechanisms that contribute to the beneficial effects of fish oil consumption are poorly understood. Mounting evidence suggests that oxidation products of EPA and DHA may be responsible, at least in part, for these benefits. Previously, we have defined the free radical-induced oxidation of arachidonic acid in vitro and in vivo and have proposed a unified mechanism for its peroxidation. We hypothesize that the oxidation of EPA can be rationally defined but would be predicted to be significantly more complex than arachidonate because of the fact that EPA contains an addition carbon-carbon double bond. Herein, we present, for the first time, a unified mechanism for the peroxidation of EPA. Novel oxidation products were identified employing state-of-the-art mass spectrometric techniques including Ag(+) coordination ionspray and atmospheric pressure chemical ionization mass spectrometry. Predicted compounds detected both in vitro and in vivo included monocylic peroxides, serial cyclic peroxides, bicyclic endoperoxides, and dioxolane-endoperoxides. Systematic study of the peroxidation of EPA provides the basis to examine the role of specific oxidation products as mediators of the biological effects of fish oil.

Electron capture atmospheric pressure photoionization mass spectrometry: analysis of fullerenes, perfluorinated compounds, and pentafluorobenzyl derivatives

An electron capture (EC) ionization mechanism has been found to be highly efficient in negative-ion atmospheric pressure photoionization (APPI) for the analysis of compounds with positive electron affinity (EA). Using negative-ion APPI, we first report the sensitive detection of natural electrophores with limited polarity, such as fullerenes and perfluorinated compounds, by mass spectrometry (MS). Using direct infusion on a quadrupole time-of-flight (QTOF) mass spectrometer, the limits of detection (LODs) for C(60) and perfluoromethylcyclohexane were determined to be 0.15 pg (0.2 fmol) and 1 femtoliter (fL) ( approximately 1.5 pg or 4.3 fmol), respectively. As the EA of the analyte increases, the detection sensitivity is enhanced. Making use of the accurate mass measurement capability of the QTOF mass spectrometer, we were able to investigate the elemental composition of the ions in each spectrum and attribute the observed high sensitivity to an EC-initiated ionization process. The proposed EC ionization mechanism is further supported by the observation of a dissociative EC reaction of pentafluorobenzyl (PFB)-derivatized phenols. The analysis of phenols by EC-APPI of their PFB derivatives resulted in very high sensitivity, with the lowest reported LOD of approximately 0.17 pg (0.5 fmol) being for 2,4-dinitrophenol. For future LC/EC-APPI-MS applications, the effect of additives and solvents on sensitivity was also tested and reported.

9- and 10-Nitro-oleic acid do not interfere with the GC–MS quantitative determination of nitrite and nitrate in biological fluids when measured as their pentafluorobenzyl derivatives

The nitrated lipids 9-nitro-oleic acid (9-NO 2-OA) and 10-nitro-oleic acid (10 -NO 2-OA) have been reported to be present in blood of healthy humans. Free and esterified forms of 9-NO 2-OA and 10-NO 2-OA have been detected in human plasma at about 600 and 300 nM, respectively. These concentrations are of the same order of magnitude of circulating nitrite. In theory, 9-NO 2-OA and 10-NO 2-OA may interfere with the analysis of circulating nitrite and nitrate. In the present study, we investigated a possible interference of 9-NO 2-OA and 10-NO 2-OA with the GC–MS method of analysis of nitrite and nitrate involving derivatization by pentafluorobenzyl (PFB) bromide in aqueous acetone at 50 °C for 5 min (nitrite) or for 60 min (nitrite and nitrate). Our results show that 9-NO 2-OA and 10-NO 2-OA do not interfere with the GC–MS analysis of nitrite and nitrate as PFB derivatives in plasma and phosphate buffered saline when added to these matrices at supraphysiological concentrations of 1–10 μM. Thus, nitrated lipids such as 9-NO 2-OA and 10-NO 2-OA can be excluded as potential interfering substances in the GC–MS quantitative determination of nitrite and nitrate as their PFB derivatives.

Lipidomic Analysis for Lipid Peroxidation-Derived Aldehydes Using Gas Chromatography-Mass Spectrometry

A lipidomic method is described for the measurement of lipid peroxidation-derived aldehydes using gas chromatography/electron ionization/mass spectrometry with selected ion monitoring (GC/EI/MS-SIM). Aldehydes in the samples were converted into their pentafluorobenzyl (PFB)-oximes using PFB-hydroxylamine, and other functional groups such as the hydroxyl groups were further derivatized into the trimethylsilyl ethers. The PFB-oxime derivatives could be comprehensively detected by the SIM of m/z 181, which is a characteristic fragment ion of the PFB-oxime derivatives. At the same time, each aldehyde was classified into five groups (alkanals, 2-alkenals, 2,4-alkadienals, 2-hydroxyalkanals, and 4-hydroxy-2-alkenals) by SIM of the structure-specific fragment ions. Determination of the 4-hydroxy-2-alkenals was also performed by confirmation of their higher reactivity with the sulfhydryl group. On the basis of the mass spectrometric characterization, we have identified at least 33 aldehydes formed upon the FeII-mediated decomposition of the arachidonic acid-, linoleic acid-, and docosahexaenoic acid-hydroperoxides in vitro. We then applied this system to the in vivo samples and successfully observed the increase in aldehydes in the liver of mice intraperitoneally injected with bromobenzene, an experimental animal model for lipid peroxidation. Using this comprehensive analysis, unique differences in the formation between each aldehyde could be observed. This method is useful for simultaneously monitoring the lipid peroxidation-derived aldehydes formed under oxidative stress in vivo.

Simultaneous Determination of 13 Phenoxy Acid Herbicide Residues in Soybean by GC‐ECD

In this study, a gas chromatography method for the multiresidue determination of 13 phenoxy acid herbicide residues (4‐CPA, 4‐CPP, phenoxy butyric acid, dicamba, MCPA, MCPP, MCPB, 2,4‐D, 3,4‐D, 2,4,5‐T, 2,4,5‐TP, 2,4‐DP, and 2,4‐DB) in soybean was developed. After the removal of fat using n‐hexane, the sample was extracted with acetonitrile‐50 mM HCl (v/v 7:3), and then followed by liquid‐liquid partition with n‐hexane (saturated by acetonitrile). The soybean extract was further cleaned up by anion exchange column. Then the residues were derived with pentafluorobenzyl bromide and the resulting pentafluorobenzyl (PFB) esters were analyzed by a gas chromatograph equipped with an electron capture detector (ECD), quantified by the external standard method. Recoveries of spiked samples at two fortified levels (0.01 and 0.1 mg/kg) are all above 70%. And the relative standard deviation was less than 20%. It shows that the limits of determination (LOD) of this method (S/N≥3) can meet the requirement of maximum residue analysis in import/export inspection for soybean.

GC‐MS analysis of aldehydes reveals diverse pathways of lipid hydroperoxide metabolism with myoglobin

A comprehensive profile of aldehyde production is being developed as a tool to probe the factors that influence lipid hydroperoxide reactions by hemeproteins. Cryofocusing coupled with GC-TOFMS provided a real-time method for monitoring the volatile metabolite hexanal, and formation of pentafluoro benzyl oxime or pentafluro phenyl hydrazine derivatives and GC analysis were used to monitor malondialdehyde (MDA) and 4hydroxynonenal (4HNE) production. Studies with equine metmyoglobin (metMb) indicate that hydroperoxides in arachidonic acid (AA) are required for MDA and 4HNE production, that the molar ratio of MDA to 4HNE formed is greater for AA than for AA in phospholipid, and that trace metals in the metMb preparations contributed to some extent to the formation of MDA and 4HNE. The results suggest that the form of AA presented to metMb influences disposition of lipid hydroperoxides. Treatment of metMb with H2O2 forms a protein-bound heme adduct (MbH). MbH is more effective than Mb at generating TBARS products with phospholipids containing AA, although much less capable of forming MDA, 4HNE, and hexanal. MbH has optimal activity at low ionic strength (0.2 mM NaOAc, pH 6 or 7), whereas Mb has optimal activity at 2 mM NaOAc, pH 5. MbH is very active at supporting formation of lipid hydroperoxides, but does not influence as extensively as does metMb their conversion to aldehydes. Supported by NSF grant 9987063.