Mass Spectrometric Quantification Research Articles

Liquid chromatography/tandem mass spectrometric quantification with metabolite screening as a strategy to enhance the early drug discovery process.

Throughput for early discovery drug metabolism studies can be increased with the concomitant acquisition of metabolite screening information and quantitative analysis using ultra-fast gradient chromatographic methods. Typical ultra-fast high-performance liquid chromatography (HPLC) parameters used during early discovery pharmacokinetic (PK) studies, for example, employ full-linear gradients over 1-2 min at very high flow rates (1.5-2 mL/min) on very short HPLC columns (2 x 20 mm). These conditions increase sample throughput by reducing analytical run time without sacrificing chromatographic integrity and may be used to analyze samples generated from a variety of in vitro and in vivo studies. This approach allows acquisition of more information about a lead candidate while maintaining rapid analytical turn-around time. Some examples of this approach are discussed in further detail.

Mass spectrometric quantification of F2-isoprostanes as indicators of oxidant stress.

Mass spectrometric quantification of F2-isoprostanes as indicators of oxidant stress.

Detection of aflatoxins (G(1-2), B(1-2)), sterigmatocystin, citrinine and ochratoxin A in samples contaminated by microbes.

A method is described for the simultaneous determination of common aflatoxins (G1, G2, B1, B2) and their precursor sterigmatocystin, and also citrinine and ochratoxin A. The method was applied to a building material matrix artificially contaminated with mycotoxin-producing fungi. The method includes extraction, sample pre-treatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionisation on a quadrupole ion trap mass analyser (ESI-MS-MS). Aqueous methanol was used in the initial extraction and solvent partitioning and solid phase extraction in the purification of samples. The HPLC separation was run on-line with the ESI-MS-MS detection. The limit of quantification of the procedure was 200 ng for all compounds. Recoveries of the sample pre-treatment varied from 28 to 99%. The average compound- and concentration-dependent accuracy and precision (RSD) were 21 and 113%, respectively. The method includes small sample volumes (approximately 1 g in 20 ml) and few, non-labour intensive, sample treatment steps. It should allow for a high throughput of samples with good prospects of automation.

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19‐norandrosterone and 19‐noretiocholanolone in human urine

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19‐norandrosterone and 19‐noretiocholanolone in human urine

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19-norandrosterone and 19-noretiocholanolone in human urine.

For the first time in the field of steroid residues in humans, demonstration of 19-norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone (19-NE: 3alpha-hydroxy-5beta-estran-17-one) excretion in urine subsequent to boar consumption is reported. Three male volunteers agreed to consume 310 g of tissues from the edible parts (meat, liver, heart and kidney) of a boar. The three individuals delivered urine samples before and during 24 h after meal intake. After deconjugation of phase II metabolites, purification and specific derivatisation of target metabolites, the urinary extracts were analysed by mass spectrometry. Identification was carried out using measurements obtained by gas chromatography/high resolution mass spectrometry (GC/HRMS) (R = 7000) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (positive electrospray ionisation (ESI+)). Quantification was realised using a quadrupole mass filter. 19-NA and 19-NE concentrations in urine reached 3.1 to 7.5 microg/L nearby 10 hours after boar tissue consumption. Levels returned to endogenous values 24 hours after. These two steroids are usually exploited to confirm the exogenous administration of 19-nortestosterone (19-NT: 17beta-hydroxyestr-4-en-3-one), especially in the antidoping field. We have thus proved that eating tissues of non-castrated male pork (in which 17beta-nandrolone is present) might induce some false accusations of the abuse of nandrolone in antidoping.

Analysis of Nicotine, 3-Hydroxycotinine, Cotinine, and Caffeine in Urine of Passive Smokers by HPLC-Tandem Mass Spectrometry

A method is described for the simultaneous analysis of nicotine and two of its major metabolites, cotinine and 3-hydroxycotinine, as well as for caffeine from urine samples. The method was developed to assess exposure of restaurant and hotel workers to environmental tobacco smoke. The method includes sample pretreatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyzer. Sample pretreatment followed standard protocols, including addition of base before liquid-liquid partitioning against dichloromethane on a solid matrix, evaporation of the organic solvent using gaseous nitrogen, and transferring to HPLC vials using HPLC buffer. HPLC separation was run on-line with the electrospray ionization-tandem mass spectrometric detection. The detection limits of the procedure were in the 1 microg/L range, except for nicotine (10 microg/L of urine). Still lower detection limits can be achieved with larger sample volumes. Recoveries of the sample treatment varied from 99% (cotinine) to 78% (3-hydroxycotinine). The method described is straightforward and not labor-intensive and, therefore, permits a high throughput of samples with excellent prospects for automation. The applicability of the method was demonstrated in a small-scale study on restaurant employees.

Mass spectrometric (HPLC/ESI–MS/MS) quantification of pyrimido[1,2-a]purin-10(3 H)-one, a guanine adduct formed by reaction of malondialdehyde with DNA

A high performance liquid chromatography/electrospray ionization–tandem mass spectrometric (HPLC/ESI–MS/MS) method has been developed for quantification of pyrimido[1,2-a]purin-10(3 H)-one adducts from DNA. The method is based on acid-catalyzed cleavage of the adducts from DNA and the use of [2,3a,10- 13C 3]pyrimido[1,2-a]purin-10(3 H)-one as an internal standard in the analysis. For this purpose the latter compound was prepared. Rate constants for the acidcatalyzed cleavage of pyrimido[1,2-a]purin-10(3 H)-one from the corresponding 2′-deoxyribonucleoside were determined, and its hydrolytic stability and possible formation by a cross reaction between guanine and [2,3a,10]pyrimido[1,2-a]purin-10(3 H)-one were studied.

Mass Spectrometric Quantification of 3-Nitrotyrosine, ortho-Tyrosine, and o,o′-Dityrosine in Brain Tissue of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated Mice, a Model of Oxidative Stress in Parkinson's Disease

Oxidative stress is implicated in the death of dopaminergic neurons in Parkinson's disease and in the 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) model of Parkinson's disease. Oxidative species that might mediate this damage include hydroxyl radical, tyrosyl radical, or reactive nitrogen species such as peroxynitrite. In mice, we showed that MPTP markedly increased levels of o, o'-dityrosine and 3-nitrotyrosine in the striatum and midbrain but not in brain regions resistant to MPTP. These two stable compounds indicate that tyrosyl radical and reactive nitrogen species have attacked tyrosine residues. In contrast, MPTP failed to alter levels of ortho-tyrosine in any brain region we studied. This marker accumulates when hydroxyl radical oxidizes protein-bound phenylalanine residues. We also showed that treating whole-brain proteins with hydroxyl radical markedly increased levels of ortho-tyrosine in vitro. Under identical conditions, tyrosyl radical, produced by the heme protein myeloperoxidase, selectively increased levels of o,o'-dityrosine, whereas peroxynitrite increased levels of 3-nitrotyrosine and, to a lesser extent, of ortho-tyrosine. These in vivo and in vitro findings implicate reactive nitrogen species and tyrosyl radical in MPTP neurotoxicity but argue against a deleterious role for hydroxyl radical in this model. They also show that reactive nitrogen species and tyrosyl radical (and consequently protein oxidation) represent an early and previously unidentified biochemical event in MPTP-induced brain injury. This finding may be significant for understanding the pathogenesis of Parkinson's disease and developing neuroprotective therapies.

Gas Chromatographic–Tandem Mass Spectrometric Quantification of Free 3-Nitrotyrosine in Human Plasma at the Basal State

A fully validated gas chromatographic–tandem mass spectrometric (GC–tandem MS) method for the accurate and precise quantification of free 3-nitrotyrosine in human plasma at the basal state is described. In the plasma of 11 healthy humans a mean concentration of 2.8 nM (range 1.4–4.2 nM) for free 3-nitrotyrosine was determined by this method. This is the lowest concentration reported for free 3-nitrotyrosine in plasma of healthy humans. The presence of endogenous free 3-nitrotyrosine in human plasma was unequivocally shown by generating a daughter mass spectrum. Various precautions had to be taken to avoid artifactual formation of 3-nitrotyrosine from nitrate during sample treatment. Endogenous plasma 3-nitrotyrosine and 3-nitro-l-[2H3]tyrosine added for use as internal standard were isolated by high-performance liquid chromatographic (HPLC) analysis of 200-μl aliquots of plasma ultrafiltrate samples (20 kDa cut-off), extracted from a single HPLC fraction by solid-phase extraction, derivatized to their n-propyl ester–pentafluoropropionyl amide–trimethylsilyl ether derivatives, and quantified by GC–tandem MS. Overall recovery was determined as 50 ± 5% using 3-nitro-l-[14C9]tyrosine. The limit of detection of the method was 4 amol of 3-nitrotyrosine, while the limit of quantitation was 125 pM using 3-nitro-l-[14C9]tyrosine. 3-Nitrotyrosine added to human plasma at 1 nM was quantitated with an accuracy of ≥80% and a precision of ≥94%. The method should be useful to investigate the utility of plasma free 3-nitrotyrosine as an indicator of nitric oxide (•NO)-associated oxidative stress in vivo in humans.

Formation of nitric oxide-derived oxidants by myeloperoxidase in monocytes: pathways for monocyte-mediated protein nitration and lipid peroxidation In vivo.

Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reaction pathways for these events in vivo are established. In the present study, we examined the chemical pathways available to monocytes for generating reactive nitrogen species and explored their potential contribution to the protein nitration and lipid peroxidation of biological targets. Isolated human monocytes activated in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide ((*)NO) metabolism, nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid peroxidation. LDL nitration (assessed by gas chromatography-mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-performance liquid chromatography with online tandem mass spectrometric quantification of distinct products) required cell activation and NO(2)(-); occurred in the presence of metal chelators, superoxide dismutase (SOD), and scavengers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibitors and catalase. Monocytes activated in the presence of the exogenous (*)NO generator PAPA NONOate (Z-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2- diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of (*)NO flux, both protein nitration and lipid peroxidation were inhibited by catalase and peroxidase inhibitors but not SOD, suggesting a role for MPO. As rates of (*)NO flux increased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadieno ic acid production by monocytes became insensitive to the presence of catalase or peroxidase inhibitors, but they were increasingly inhibited by SOD and methionine, suggesting a role for peroxynitrite. Collectively, these results demonstrate that monocytes use distinct mechanisms for generating (*)NO-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.

Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS.

Genistein is the principal soy isoflavone to which the putative beneficial effects of soy consumption have been attributed; however, the possibility of adverse biological effects (e.g., estrogenic, antithyroid) has also been raised. This paper describes development and validation of a simple and sensitive analytical method for the determination of genistein in the blood of rats receiving dietary genistein (<0.5-1250 microg of genistein aglycone/g of chow). The method uses serum/plasma deproteination, liquid-liquid extraction, deuterated genistein and daidzein internal standards, isocratic LC separation, and electrospray mass spectrometric quantification using selected ion monitoring. Extraction efficiency is approximately 85%, the detection limits for genistein and daidzein from 50 microL of rat blood are approximately 5 nM, and the limit of quantification is approximately 15 nM. Interassay precision (relative standard deviation 4.5-4.6%) and intraassay precision (3.3-6.7%) were determined from replicate analysis of a spiked control and an incurred serum sample. The distribution of conjugated and unconjugated forms of genistein in the blood of rats was determined using selective enzyme hydrolysis. The glucuronide was the predominant metabolite (>90%), and only small amounts of the sulfate conjugate and the aglycone were observed at all dose levels. No evidence for additional metabolites was obtained. The 7- and 4'-glucuronide conjugates of genistein were identified using electrospray mass spectrometry and (1)H NMR. Total blood genistein ranged from <15 nM in animals fed soy-free control diet to as high as 8.9 microM in male rats fed 1250 microg of genistein/g of chow and encompasses blood isoflavone levels observed in humans consuming a typical Asian diet and nutritional supplements (0.1-1 microM) and infants consuming soy formulas (2-7 microM).

Gas chromatographic–tandem mass spectrometric quantification of human plasma and urinary nitrate after its reduction to nitrite and derivatization to the pentafluorobenzyl derivative

Gas chromatography–mass spectrometry (GC–MS) of nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode is a useful analytical tool to quantify accurately and sensitively nitrite and nitrate after its reduction to nitrite in various biological fluids. In the present study we demonstrate the utility of GC–tandem MS to quantify nitrate in human plasma and urine. Our present results verify human plasma and urine levels of nitrite and nitrate measured previously by GC–MS.

Mass spectrometric quantification of amino acid oxidation products in proteins: insights into pathways that promote LDL oxidation in the human artery wall.

Oxidatively damaged low density lipoprotein (LDL) may play an important role in atherogenesis, but the physiologically relevant pathways have proved difficult to identify. Mass spectrometric quantification of stable compounds that result from specific oxidation reactions represents a powerful approach for investigating such mechanisms. Analysis of protein oxidation products isolated from atherosclerotic lesions implicates tyrosyl radical, reactive nitrogen species, and hypochlorous acid in LDL oxidation in the human artery wall. These observations provide chemical evidence for the reaction pathways that promote LDL oxidation and lesion formation in vivo.--Heinecke, J. W. Mass spectrometric quantification of amino acid oxidation products in proteins: insights into pathways that promote LDL oxidation in the human artery wall.

Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress

This chapter has outlined methods to assess lipid peroxidation associated with oxidant injury in vivo by quantifying concentrations of free F2-IsoPs in biological fluids and levels of F2-IsoPs esterified in tissue lipids. The mass spectrometric assay described herein is highly precise and accurate. A potential shortcoming with this approach is that it requires expensive instrumentation, i.e., a mass spectrometer. However, several immunoassays for an F2-IsoP, 8-iso-PGF2 alpha, have become available from commercial sources. At this time, the accuracy and reliability of these assay for quantifying F2-IsoPs in biological fluids has not been fully validated by mass spectrometry. If these immunoassays prove to be a reliable measure of F2-IsoPs, however, this should greatly expand the use of F2-IsoPs to assess oxidant stress. In conclusion, studies carried out over the past several years have shown that measurement of F2-IsoPs has overcome many of the limitations associated with other methods to assess oxidant status, especially when applied to the measurement of oxidant stress in vivo in humans. Therefore, the quantification of F2-IsoPs represents an important advance in our ability to assess the role of oxidant stress and lipid peroxidation in human disease.

Gas chromatographic–mass spectrometric identification and quantification of aniline after extraction from serum and derivatization with 2,2,2-trichloroethyl chloroformate, a novel derivative

Aniline is widely used as an intermediate in the synthesis of dyes. It is also used in the manufacture of pharmaceuticals, photographic developers, shoe polish, etc. Exposure to aniline is toxic because it produces methemoglobin. In humans, blood methemoglobin levels are often measured as an index of exposure to aniline. Here a method is described for identification and quantification of aniline by gas chromatography–mass spectrometry after extraction from human serum and derivatization with 2,2,2-trichloroethyl chloroformate. Aniline, along with the internal standard N-methylaniline, were extracted from alkaline serum using chloroform. Aniline and the internal standard were derivatized with 50 μl 2,2,2-trichloroethyl chloroformate. After evaporating excess derivatizing reagent, the residue was reconstituted in 50 μl chloroform and injected into the gas chromatographic–mass spectrometry (GC–MS) system. A positive identification of derivatized aniline can be made by observing strong molecular ions at m/ z 267 and 269. Similarly, the derivatized internal standard showed strong molecular ions at m/ z 281 and 283. The within-run and between-run precisions of the assay were 3.61 and 5.92%, respectively, at an aniline concentration of 5 mg/l. The assay was linear for serum aniline concentrations of 0.5–25.0 mg/l. The detection limit was 0.1 mg/l. The assay was not affected by lipemia, hemolysis or high bilirubin concentration in serum.

Isotope Dilution Mass Spectrometric Quantification of 3-Nitrotyrosine in Proteins and Tissues Is Facilitated by Reduction to 3-Aminotyrosine

Oxidative damage by reactive nitrogen species has been implicated in the pathogenesis of atherosclerosis and other inflammatory diseases. The mechanisms of tissue damage are poorly understood, however, because the toxic intermediates are short-lived. Previousin vitrostudies have suggested that 3-nitrotyrosine represents a specific marker of protein oxidation by reactive nitrogen species. The detection of this nitrated aromatic amino acid may thus serve as an indicator of tissue injury by nitrogen speciesin vivo.Here we describe a highly sensitive and specific analytical method for quantifying free and protein-bound 3-nitrotyrosine. The assay involves acid hydrolysis of proteins, isolation of 3-nitrotyrosine by ion exchange chromatography, and reduction of 3-nitrotyrosine to 3-aminotyrosine with dithionite. The reduced amino acid is then converted to itsn-propyl, per-heptafluorobutyryl derivative and quantified by isotope dilution gas chromatography negative-ion chemical ionization mass spectrometry. Attomole levels of 3-nitrotyrosine can be reproducibly measured in this manner. Quantifying 3-nitrotyrosine levels of tissues by stable isotope dilution gas chromatography/mass spectrometry should provide a powerful tool for exploring the impact of reactive nitrogen species on oxidative reactionsin vivo.

Detection of polar and macrocyclic trichothecene mycotoxins from indoor environments.

A method is described for the qualitative and semi-quantitative simultaneous determination of both non-macrocyclic and macrocyclic trichothecene biotoxins from samples derived from indoor environments. The method includes extraction, sample pre-treatment and reversed-phase HPLC separation followed by tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyser. Aqueous methanol was used in the initial extraction and solvent partitioning and solid-phase extraction in the purification of samples. The HPLC separation was run on-line with electrospray ionization MS-MS detection. The detection limits and recoveries of the procedure varied from 1 to 1000 pg and from 31 to 92%, respectively. As the method includes few and not very labour intensive sample treatment steps, it should allow for a high throughput of samples with good prospects of automation.

Mass spectrometric quantification of the mu opioid receptor agonist Tyr- d-Arg-Phe-Lys-NH 2 (DALDA) in high-performance liquid chromatography-purified ovine plasma

The mu opioid receptor agonist Tyr- d-Arg-Phe-Lys-Amide ( d - Arg 2- Lys 4- Dermorphin 1-4 amide=DALDA) was infused continuously for 2 h into sheep. The presence of DALDA in ovine plasma was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) in plasma samples that were obtained at different times during and following that infusion. A stable isotope-incorporated internal standard, deuterated DALDA (d 5-DALDA), was used for the MS quantification of DALDA via the protonated molecule ion, (M+H) +, of DALDA and of d 5-DALDA. Time-course data (μg DALDA ml −1 plasma vs. time) were obtained. Tandem MS (MS–MS) provided the product-ion spectrum of the (M+H) + ion of DALDA in one of the samples to confirm the amino acid sequence of DALDA.

Simultaneous solid phase extraction, derivatization, and gas chromatographic mass spectrometric quantification of thromboxane and prostacyclin metabolites, prostaglandins, and isoprostanes in urine.

The current analytical methods for the various prostanoids require a separate and extended sample workup, derivatization, and gas chromatographic/mass spectrometric detection of each compound. Therefore, we developed and validated a rapid method for the common purification, derivatization, and GC/MS determination of 11-dehydrothromboxane B2, 2,3-dinor-6-keto-PGF1a, PGF2A, PGE2, PGD2, and isoprostanes in urine. A single reversed-phase solid-phase extraction step and modified reaction conditions yielded excellent sample purification at high recoveries and efficient derivatization for all compounds in one vial. The method allows, for the first time, the simultaneous quantification of these index metabolites of systemic thromboxane and prostacyclin synthesis, renal prostaglandin formation, and nonenzymatic in vivo lipid peroxidation in a single GC/MS run with high sensitivity and precision.

Mass spectrometric identification and quantification of hemorphins extracted from human adrenal and pheochromocytoma tissue.

The hemorphins are a family of recently identified opioid receptor binding peptides derived from the proteolytic processing of the beta, gamma, delta, and epsilon chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocytoma tumors. The hemorphins were quantified and structurally characterized using mass spectrometry. High concentrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue.