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Analytical and Bioanalytical Chemistry (v.403, #7)

JaVed I. Khan, Thomas J. Kennedy, and Donnell R. Christian Jr.: Basic principles of forensic chemistry by Pascal Kintz (pp. 1773-1774).

Biomedical mass spectrometry by Toyofumi Nakanishi; Mitsutoshi Setou; Tomiko Kuhara (pp. 1775-1776).

is an associate professor of clinical and laboratory medicine at Osaka Medical College. His research interests include clinical application of imaging mass spectrometry and clinical diagnosis of soft-ionization mass spectrometry coupled with immunoprecipitation. He is the author of 65 peer-reviewed papers, 35 scientific papers, and 13 book chapters. has been a full professor of anatomy and cell biology at the Hamamatsu University School of Medicine since 2008. His research interests include development and application of imaging mass spectrometry and systems biology with “omics” technologies. He is the author of over 100 peer-reviewed papers, 150 scientific papers, and ten book chapters, and is the editor of a book on imaging mass spectrometry. president of JSBMS from 2007 to 2011, is a professor of human genetics at Kanazawa Medical University. Her research interests have included the development and application of metabolic profiling based on gas chromatography/mass spectrometry since the 1970s and metabolomic profiling since the early 1990s. The diagnostic procedures she has developed are extremely useful for noninvasive differential chemical diagnosis of around 130 disease conditions (including more than 100 inherited metabolic disorders) and for personalized medicine. The procedures are also very effective for all stages of drug R & D.

Posttranslational modification of indoleamine 2,3-dioxygenase by Hidetsugu Fujigaki; Mitsuru Seishima; Kuniaki Saito (pp. 1777-1782).

Protein posttranslational modifications (PTMs) perform essential roles in the biological regulation of a cell. PTMs are extremely important because they can change a protein’s physical or chemical properties, conformation, activity, cellular location, or stability. In fact, most proteins are altered by the addition or removal of a chemical moiety on either an amino acid or the protein’s N- or C-terminus. Some PTMs can be added and removed dynamically as a mechanism for reversibly controlling protein function. Thus, identifying the PTM sites is critical to fully understand the biological roles of any given protein. Mass spectrometry (MS) is a widely used analytical strategy to identify PTMs. We have used an automated two-dimensional liquid chromatography (LC) system coupled with electrospray ionization quadrupole ion-trap MS to identify PTMs for indoleamine 2,3-dioxygenase 1 (IDO1), one of the tryptophan catabolic enzymes. IDO1 promotes immune tolerance by suppressing local T-cell responses under various physiological and pathophysiological conditions, such as pregnancy in mammals, tumor resistance, autoimmunity, and chronic inflammation. Although many studies have demonstrated the biological importance of IDO activity, the PTMs of IDO enzymes remain largely unknown. Only a few important PTMs of IDO1 have been found, such as nitration, N-terminal acetylation, and phosphorylation. In this review, we analyze the PTMs of IDO1 using our two-dimensional LC-MS/MS system, and provide an overview of our current understanding.

Keywords: Indoleamine 2,3-dioxygenase; Posttranslational modification; l-Tryptophan; Kynurenine

Microbial degradation of physiologically active peptides by strain B-9 by Fumio Kondo; Shoshiro Okada; Atsushi Miyachi; Miki Kurita; Kiyomi Tsuji; Ken-ichi Harada (pp. 1783-1791).

The reaction of some physiologically active peptides with bacterial strain B-9 has been investigated. Bradykinin, β-endorphin, and [Leu⁵]enkephalin were quickly degraded, with half-lives of <5 min. Somatostatin, substance P, and angiotensin I were degraded relatively smoothly, with half-lives of 10 min to 1 h, whereas oxytocin and insulin were slowly degraded, with half-lives of 1 and 4 days, respectively. Vasopressin was barely degraded, with a half-life of >7 days. Linearized vasopressin, prepared by the reductive cleavage of the disulfide bond followed by alkylation with iodoacetamide, was degraded significantly faster than intact vasopressin, with a half-life of 2.5 h. A loop formed by disulfide bond formation was regarded as one of the degradation-resistant factors. Hydrolysis of the peptides in this study took place through cleavage of various peptide bonds, and the strain B-9 may bear similarities to the neutral endopeptidase in terms of its broad selectivity.

Keywords: Strain B-9; Biodegradation; Physiologically active peptides

Determination of azide in gastric fluid and urine by flow-injection electrospray ionization tandem mass spectrometry by Kayoko Minakata; Hideki Nozawa; Itaru Yamagishi; Kunio Gonmori; Koutaro Hasegawa; Masako Suzuki; Kanako Watanabe; Osamu Suzuki (pp. 1793-1799).

A rapid method was developed to identify and quantify the azide ion (N₃ ^–) in gastric fluid and urine. N₃ ^– in diluted biological fluids was reacted with NaAuCl₄ to produce Au(N₃)₂ ^–, which was extracted with octanol. Five microliters of the extract were flow-injected into an electrospray ionization tandem mass spectrometric instrument. Quantification of N₃ ^– was performed by selected reaction monitoring of the product ion Au(N)(N₃) ^– at m/z 253, which was derived from the precursor ion Au(N₃)₂ ^– at m/z 281, using 50 μL of aqueous solution within 10 min. This method was found to be linear up to 10^–5 M, to have a limit of quantification of 10^–7 M, a limit of detection of 3.0 × 10^–8 M, and a coefficient of variation of ≦10% at 10^–7 M. In the case of urine, 50 μL of urine were spiked with N₃ ^–, this was diluted 10-fold and passed through 1 mL of a resin, and finally diluted to 100-fold of the original. This method was linear up to 10^–3 M, had a limit of quantification of 10^–5 M, a limit of detection of 3.0 × 10^–6 M, and coefficient of variation of ≦8.8% for an original urine concentration of 10^–5 M. The practical applicability of this method was checked by diluting 1 μL of a suspected suicide victim’s gastric fluid 20,000-fold and 1 μL of the victim’s urine 5,000-fold and then measuring the N₃ ^– levels. These levels were found to be (7.5 ± 1.0) × 10^–2 M and (3.2 ± 0.4) × 10^–3 M, respectively. Figure The product ions were observed at m/z 253, 225 and 42 for azide but that was observed only at m/z 42 for OCN.

Keywords: Azide; Gold; Electrospray ionization; Tandem mass spectrometry; Gastric fluid; Urine

Identification of β-tubulin as a common immunogen in gastrointestinal malignancy by mass spectrometry of colorectal cancer proteome: implications for early disease detection by George Bouras; Toyofumi Nakanishi; Yoshihisa Fujita; Soichiro Tsunemi; Takayuki Takubo; Nobuhiko Tanigawa (pp. 1801-1809).

Rapid identification of microorganisms by mass spectrometry: improved performance by incorporation of in-house spectral data into a commercial database by Kazuyuki Sogawa; Masaharu Watanabe; Kenichi Sato; Syunsuke Segawa; Akiko Miyabe; Syota Murata; Tomoko Saito; Fumio Nomura (pp. 1811-1822).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used as a microbial diagnostic method for species identification of pathogens. However, MALDI-TOF identification of bacteria at the species level remains unsatisfactory, with the major problem being an incomplete database that still needs refinement and expansion. Augmentation of the original MALDI BioTyper 2.0 (Bruker) database by incorporating mass spectra obtained in-house from clinical isolates may increase the identification rate at the species level. We conducted a prospective study to assess whether the augmented database can improve the performance of MALDI-TOF MS for routine identification of species. Cluster analyses revealed distinct differences in MS spectral profiles of clinical isolates obtained in our hospital and those of ATCC strains in the Bruker database. In the first part of the study, which was performed over 3 weeks, 259 bacterial isolates were subjected to analysis by MALDI-TOF MS, and MS spectra of 229 successfully identified isolates (49 species) were incorporated into the original database to give the augmented Bruker–Chiba database. In a second separate analysis, the concordance of identification of 498 clinical isolates of the 49 species with conventional methods was 87.1% (434/498) with the commercial Bruker database and 98.0% (488/498) using the Bruker–Chiba database. These results indicate that refinement of a commercial database can be achieved relatively easy and effectively by incorporating MS spectra of clinical isolates obtained in a clinical laboratory.

Keywords: Clinical isolate; In-house database; MALDI BioTyper software; MALDI-TOF MS

Development and validation of a GC-EI-MS method with reduced adsorption loss for the quantification of olanzapine in human plasma by Kayo Ikeda; Kazuro Ikawa; Toshihiro Kozumi; Satoko Yokoshige; Shunji Horikawa; Norifumi Morikawa (pp. 1823-1830).

A simple and sensitive GC-EI-MS method using solvent extraction and evaporation was developed for the determination of olanzapine concentrations in plasma samples. Because olanzapine and promazine, which was used as the internal standard (IS), are nitrogenous bases, they can adsorb to the weakly acidic silanol groups on the surfaces of glass centrifuge tubes during solvent extraction and evaporation. Silylation of the glass tubes, addition of triethylamine (TEA), and use of a sample solution with a basic pH could prevent adsorption loss. The extraction method involved mixing plasma (500 μL) in a silylated glass tube with a promazine solution (2 μg/mL, 25 μL) in methanol containing 1% TEA. After addition of aqueous sodium carbonate (0.5 mol/L, pH 11.1, 1 mL) and extraction into 3 mL of dichloromethane/n-hexane (1:1, v/v) containing 1% TEA, the organic phase was evaporated to dryness in a silylated glass tube. The residue was dissolved in ethyl acetate containing 1% TEA (50 μL). For GC-EI-MS analysis, the calibration curves of olanzapine in human plasma were linear from 0.5 to 100 ng/mL. Intra- and interday precisions in plasma were both less than 7.36% (coefficient of variation), and the accuracy was between 94.6 and 110% for solutions with concentrations greater than 0.5 ng/mL. The limit of quantification was 0.5 ng/mL in plasma. The assay was applied to therapeutic drug monitoring in samples from three schizophrenic patients.

Keywords: Olanzapine; GC-MS; EI; Triethylamine; Silylation; Basic pH

Quantitative determination of phosphatidylcholine hydroperoxides during copper oxidation of LDL and HDL by liquid chromatography/mass spectrometry by Shu-Ping Hui; Yudai Taguchi; Seiji Takeda; Futaba Ohkawa; Toshihiro Sakurai; Shinobu Yamaki; Shigeki Jin; Hirotoshi Fuda; Takao Kurosawa; Hitoshi Chiba (pp. 1831-1840).

1-Palmitoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 16:0/18:2-OOH) and 1-stearoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 18:0/18:2-OOH) were measured by liquid chromatography/mass spectrometry (LC/MS) using nonendogenous 1-palmitoyl-2-heptadecenoylphosphatidylcholine monohydroperoxide as an internal standard. The calibration curves for synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, which were obtained by direct injection of the internal standard into the LC/MS system, were linear throughout the calibration range (0.8–12.8 pmol). Within-day and between-day coefficients of variation were less than 10%, and the recoveries were between 86% and 105%. The limit of detection (LOD) and the limit of quantification (LOQ) were determined using synthetic standards. The LOD (signal-to-noise ratio 3:1) was 0.01 pmol, and the LOQ (signal-to-noise ratio 6:1) was 0.08 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. With use of this method, the concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH in the lipoprotein fractions during copper-mediated oxidation were determined. We prepared oxLDL and oxHDL by incubating native LDL and native HDL from human plasma (n = 10) with CuSO₄ for up to 4 h. The time course of the PC 16:0/18:2-OOH and PC 18:0/18:2-OOH levels during oxidation consisted of three phases. For oxidized LDL, both compounds exhibited a slow lag phase and a subsequent rapidly increasing propagation phase, followed by a gradually decreasing degradation phase. In contrast, for oxidized HDL, both compounds initially exhibited a prompt propagation phase with a subsequent plateau phase, followed by a rapid degradation phase. The analytical LC/MS method for phosphatidylcholine hydroperoxides might be useful for the analysis of biological samples. Online Abstract Figure Quantitative determination of phosphatidylcholine hydroperoxides during copper-oxidation of LDL and HDL by liquid chromatography/mass spectrometry

Keywords: Lipid hydroperoxide; Oxidized low-density lipoprotein; Oxidized high-density lipoprotein; Liquid chromatography; Liquid chromatography/mass spectrometry; Mass spectrometry

Protein-bound uremic toxins in hemodialysis patients measured by liquid chromatography/tandem mass spectrometry and their effects on endothelial ROS production by Yoshiharu Itoh; Atsuko Ezawa; Kaori Kikuchi; Yoshinari Tsuruta; Toshimitsu Niwa (pp. 1841-1850).

Cardiovascular disease (CVD) is prevalent in patients with chronic kidney disease (CKD). In hemodialysis (HD) patients, some protein-bound uremic toxins are considered to be associated with CVD. However, it is not yet known which uremic toxins are important in terms of endothelial toxicity. Serum samples were obtained from 45 HD patients before and after HD. Total and free serum concentrations of indoxyl sulfate, indoxyl glucuronide, indoleacetic acid, p-cresyl sulfate, p-cresyl glucuronide, phenyl sulfate, phenyl glucuronide, phenylacetic acid, phenylacetyl glutamine, hippuric acid, 4-ethylphenyl sulfate, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) were simultaneously measured by liquid chromatography/electrospray ionization–mass spectrometry/mass spectrometry (LC/ESI-MS/MS). The effects of these solutes at their pre-HD mean and maximum serum concentrations on reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVEC) were measured with a ROS probe. Serum levels of 11 of the solutes (all except 4-ethylphenyl sulfate) were significantly increased in HD patients compared to healthy subjects. All 12 solutes showed changes in their protein-binding ratios. In particular, indoxyl sulfate, p-cresyl sulfate, CMPF, and 4-ethylphenyl sulfate showed high protein-binding ratios (>95 %) and low reduction rates by HD (<35 %). Indoxyl sulfate at its mean and maximum pre-HD serum concentrations—even with 4 % albumin—stimulated ROS production in HUVEC most intensely, followed by CMPF. In conclusion, the serum levels of 11 protein-bound uremic toxins were increased in HD patients. Indoxyl sulfate, p-cresyl sulfate, and CMPF could not be removed efficiently by HD due to their high protein-binding ratios. Indoxyl sulfate most intensely induced endothelial ROS production, followed by CMPF.

Keywords: Uremic toxin; Protein binding; Indoxyl sulfate; Hemodialysis; Endothelial cell; Reactive oxygen species

Visualization of acetylcholine distribution in central nervous system tissue sections by tandem imaging mass spectrometry by Yuki Sugiura; Nobuhiro Zaima; Mitsutoshi Setou; Seiji Ito; Ikuko Yao (pp. 1851-1861).

Metabolite distribution imaging via imaging mass spectrometry (IMS) is an increasingly utilized tool in the field of neurochemistry. As most previous IMS studies analyzed the relative abundances of larger metabolite species, it is important to expand its application to smaller molecules, such as neurotransmitters. This study aimed to develop an IMS application to visualize neurotransmitter distribution in central nervous system tissue sections. Here, we raise two technical problems that must be resolved to achieve neurotransmitter imaging: (1) the lower concentrations of bioactive molecules, compared with those of membrane lipids, require higher sensitivity and/or signal-to-noise (S/N) ratios in signal detection, and (2) the molecular turnover of the neurotransmitters is rapid; thus, tissue preparation procedures should be performed carefully to minimize postmortem changes. We first evaluated intrinsic sensitivity and matrix interference using Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS) to detect six neurotransmitters and chose acetylcholine (ACh) as a model for study. Next, we examined both single MS imaging and MS/MS imaging for ACh and found that via an ion transition from m/z 146 to m/z 87 in MS/MS imaging, ACh could be visualized with a high S/N ratio. Furthermore, we found that in situ freezing method of brain samples improved IMS data quality in terms of the number of effective pixels and the image contrast (i.e., the sensitivity and dynamic range). Therefore, by addressing the aforementioned problems, we demonstrated the tissue distribution of ACh, the most suitable molecular specimen for positive ion detection by IMS, to reveal its localization in central nervous system tissues.

Keywords: Imaging mass spectrometry; Neurotransmitter; Acetylcholine; MS; MS/MS; Imaging; IMS

Visualization of dynamic change in contraction-induced lipid composition in mouse skeletal muscle by matrix-assisted laser desorption/ionization imaging mass spectrometry by Naoko Goto-Inoue; Yasuko Manabe; Shouta Miyatake; Shinya Ogino; Ai Morishita; Takahiro Hayasaka; Noritaka Masaki; Mitsutoshi Setou; Nobuharu L. Fujii (pp. 1863-1871).

Lipids in skeletal muscle play a fundamental role both in normal muscle metabolism and in disease states. Skeletal muscle lipid accumulation is associated with several chronic metabolic disorders, including obesity, insulin resistance, and type 2 diabetes. However, it is poorly understood whether the lipid composition of skeletal muscle changes by contraction, due to the complexity of lipid molecular species. In this study, we used matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to investigate changes in skeletal muscle lipid composition induced by contraction. We successfully observed the reduction of diacylglycerol and triacylglycerol, which are generally associated with muscle contraction. Interestingly, we found the accumulation of some saturated and mono-unsaturated fatty acids and poly-unsaturated fatty acids containing phosphatidylcholine in contracted muscles. Moreover, the distributions of several types of lipid were changed by contraction. Our results show that changes in the lipid amount, lipid composition, and energy metabolic activity can be evaluated in each local spot of cells and tissues at the same time using MALDI-IMS. In conclusion, MALDI-IMS is a powerful tool for studying lipid changes associated with contractions.

Keywords: Biopolymers/lipids; Imaging (NMR microscopy|electron microscopy); Mass spectrometry/ICP-MS; Biological samples

Spatiotemporal alteration of phospholipids and prostaglandins in a rat model of spinal cord injury by Mitsuru Hanada; Yuki Sugiura; Ryuichi Shinjo; Noritaka Masaki; Shiro Imagama; Naoki Ishiguro; Yukihiro Matsuyama; Mitsutoshi Setou (pp. 1873-1884).

We determined quantitative and qualitative alterations in lipids during the occurrence and progression of spinal cord injury (SCI) in rats to identify potential clinical indicators of SCI pathology. Imaging mass spectrometry (IMS) was used to visualize twelve molecular species of phosphatidylcholine (PC) on thin slices of spinal cord with SCI. In addition, twelve species of phospholipids and five species of prostaglandins (PGs) were quantified by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) of lipid extracts from control/injured spinal cords. Unique distribution patterns were observed for phospholipids with different fatty acid compositions, and distinct dynamic changes were seen in both their amounts and their distributions in tissue as tissue damage resulting from SCI progressed. In particular, PCs containing docosahexaenoic acid localized to the large nucleus in the anterior horn region at one day post-SCI and rapidly decreased thereafter. In contrast, PCs containing arachidonic acid (AA-PCs) were normally found in the posterior horn region and were intensely and temporarily elevated one week after SCI. Lysophosphatidylcholines (LPCs) also increased at the same SCI stage and in regions with elevated AA-PCs, indicating the release of AA and the production of PGs. Moreover, LC-ESI-MS/MS analysis of lipid extracts from the spinal cord tissue at the impact site demonstrated a peak in PGE2 that reflected the elevation/reduction pattern of AA-PCs and LPC. Although further investigation is required, we suggest that invasive immune cells that penetrated from the impaired blood–brain barrier at 1–2 weeks post-SCI may have produced LPCs, released AA from AA-PCs, and produced PGs in SCI tissue at sites enriched in AA-PCs/LPC.

Keywords: Imaging mass spectrometry; LC-ESI-MS/MS; Phospholipids; Prostaglandins; Spinal cord injury

Visualization of anthocyanin species in rabbiteye blueberry Vaccinium ashei by matrix-assisted laser desorption/ionization imaging mass spectrometry by Yukihiro Yoshimura; Hirofumi Enomoto; Tatsuya Moriyama; Yukio Kawamura; Mitsutoshi Setou; Nobuhiro Zaima (pp. 1885-1895).

Anthocyanins are naturally occurring compounds that impart color to fruits, vegetables, and plants, and are believed to have a number of beneficial health effects in both humans and animals. Because of these properties, pharmacokinetic analysis of anthocyanins in tissue has been performed to quantify and identify anthocyanin species although, currently, no methods exist for investigating tissue localization of anthocyanin species or for elucidating the mechanisms of anthocyanin activity. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of a wide range of biomolecules. To investigate whether anthocyanin species could be identified and visualized by IMS, we performed matrix-assisted laser desorption/ionization (MALDI)–IMS analysis, by tandem mass spectrometry (MALDI–IMS–MS), of ten anthocyanin molecular species in rabbiteye blueberry (Vaccinium ashei). The distribution patterns of each anthocyanin species were different in the exocarp and endocarp of blueberry sections. Anthocyanin species composed of delphinidin and petunidin were localized mainly in the exocarp. In contrast, those species composed of cyanidin, peonidin, and malvidin were localized in both the exocarp and the endocarp. Moreover, MALDI–IMS analysis of anthocyanidins in a blueberry section indicated that the distribution patterns of each anthocyanidin species were nearly identical with those of the corresponding anthocyanins. These results suggested that the different distribution patterns of anthocyanin species in the exocarp and endocarp depended on the aglycone rather than on the sugar moieties. This study is the first to visualize anthocyanin molecular species in fruits.

Keywords: Agriculture; Foods/beverages; Mass spectrometry/ICP–MS; Natural products

Simultaneous quantitation of sphingoid bases and their phosphates in biological samples by liquid chromatography/electrospray ionization tandem mass spectrometry by Daisuke Saigusa; Kanako Shiba; Asuka Inoue; Kotaro Hama; Michiyo Okutani; Nagisa Iida; Masayoshi Saito; Kaori Suzuki; Tohru Kaneko; Naoto Suzuki; Hiroaki Yamaguchi; Nariyasu Mano; Junichi Goto; Takanori Hishinuma; Junken Aoki; Yoshihisa Tomioka (pp. 1897-1905).

We developed a liquid chromatography/electrospray ionization tandem mass spectrometry method for the simultaneous quantitative determination of C18 sphingosine (Sph), C18 dihydrosphingosine (dhSph), C18 phytosphingosine (pSph), C18 sphingosine-1-phosphate (S1P), C18 dihydrosphingosine-1-phosphate (dhS1P), and C18 phytosphingosine-1-phosphate (pS1P). Samples were prepared by simple methanol deproteinization and analyzed in selected reaction monitoring modes. No peak tailing was observed on the chromatograms using a Capcell Pak ACR column (1.5 mm i.d. × 250 mm, 3 μm, Shiseido). The calibration curves of the sphingoids showed good linearity (r > 0.996) over the range of 0.050–5.00 pmol per injection. The accuracy and precision of this method were demonstrated using four representative biological samples (serum, brain, liver, and spleen) from mice that contained known amounts of the sphingoids. Samples of mice tissue such as plasma, brain, eye, testis, liver, kidney, lung, spleen, lymph node, and thymus were examined for their Sph, dhSph, pSph, S1P, dhS1P, and pS1P composition. The results confirmed the usefulness of this method for the physiological and pathological analysis of the composition of important sphingoids. Figure Analysis of the biological distribution of seven sphingoids and their phosphates

Keywords: Sphingoid; Sphingoid phosphate; LC/MS/MS; Simultaneous quantitation; Tissue; Plasma

Use of the HS-PTR-MS for online measurements of pyrethroids during indoor insecticide treatments by Aude Vesin; Guy Bouchoux; Etienne Quivet; Brice Temime-Roussel; Henri Wortham (pp. 1907-1921).

A high-sensitivity proton transfer reaction mass spectrometer (HS-PTR-MS) has been used to study the temporal evolution of pesticide concentrations in indoor environments. Because of the high time variability of the indoor air concentrations during household pesticide applications, the use of this online high time resolution instrument is found relevant. Four pyrethroid pesticides of the latest generation that are commonly found in electric vaporizer refills, namely, transfluthrin, empenthrin, tetramethrin, and prallethrin, were considered. A controlled pesticide generation system was settled and coupled to a HS-PTR-MS analyzer, and a calibration procedure based on the fragmentation patterns of the protonated molecules was performed. To illustrate the functionality of the method, measurements of the concentration–time profiles of transfluthrin contained in an electric vaporizer were carried out in a full-scale environmental room under air exchange rate-controlled conditions. This study demonstrates that the HS-PTR-MS technique can provide online and high time-resolved measurements of semi-volatile organic compounds such as pyrethroid insecticides.

Keywords: HS-PTR-MS; Pesticides; Indoor air quality; Gas phase; Proton affinity

Carbohydrate analysis of hemicelluloses by gas chromatography–mass spectrometry of acetylated methyl glycosides by Zsuzsa Sárossy; David Plackett; Helge Egsgaard (pp. 1923-1930).

A method based on gas chromatography–mass spectrometry analysis of acetylated methyl glycosides was developed in order to analyze monosaccharides obtained from various hemicelluloses. The derivatives of monosaccharide standards, arabinose, glucose, and xylose were studied in detail and ¹³C-labeled analogues were used for identification and quantitative analysis. Excellent chromatographic separation of the monosaccharide derivatives was found and identification of the anomeric configuration was feasible through a prepared and identified pure methyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside. The electron ionization mass spectrum and fragmentation path was studied for each monosaccharide derivative. Fragment ion pairs of labeled and unlabeled monosaccharides were used for quantification; m/z 243/248 for glucose, 128/132 for xylose, and 217/218 for arabinose. Using the intensity ratios obtained from the extracted ion chromatograms, accurate quantification of monosaccharide constituents of selected hemicelluloses was demonstrated.

Keywords: Acid methanolysis; Hemicellulose; Arabinoxylan; Stable isotope; Methyl glycoside; Gas chromatography–mass spectrometry

Liquid chromatography–mass spectrometry for C₆₀ fullerene analysis: optimisation and comparison of three ionisation techniques by Ling Li; Sami Huhtala; Markus Sillanpää; Pirjo Sainio (pp. 1931-1938).

The increasing use and production of nanomaterials have led to growing concern over the release of new pollutants to the environment. Fullerenes have been a subject of intense research, both because of their unique chemistry and because of technological applications. The development of analytical methods to quantify the fullerenes in complex sample matrices is a crucial step in the study of their occurrence and exposure, and thus in risk assessment. This paper reports the development and optimisation of a method combining liquid chromatography with ion-trap mass spectrometry (LC–ITMS) for analysis of the fullerene C₆₀. Under the optimised chromatogram conditions, a C₁₈ analytical column had good selectivity for fullerenes C₆₀ and C₇₀, with retention times of 3.0 and 4.1 min, respectively. Mass spectrometric detection was tested and optimised using three common ionisation techniques—atmospheric-pressure chemical ionisation (APCI), atmospheric-pressure photoionisation (APPI), and electrospray ionisation (ESI). The molecular ion was most abundant for C ₆₀ ⁻ (m/z = 720) in APCI and APPI, whereas adduct ions were formed with the molecular ion in ESI. Finally, the performance of the three ionisation techniques examined was compared by use of five validation criteria. The instrument detection limit (8 ng mL⁻¹), quantification limit (27 ng mL⁻¹), detection sensitivity (90.2 ng mL⁻¹), linear range (8–1,000 ng mL⁻¹), and repeatability (15 %) of APPI make it the most promising ionisation technique for fullerene C₆₀ analysis.

Keywords: C₆₀ fullerene; Atmospheric-pressure chemical ionisation (APCI); Atmospheric-pressure photoionisation (APPI); Electrospray ionisation (ESI); Ion-trap mass spectrometry; Liquid chromatography

Hydrophilic properties as a new contribution for computer-aided identification of short peptides in complex mixtures by Christelle Harscoat-Schiavo; Claudia Nioi; Evelyne Ronat-Heit; Cédric Paris; Régis Vanderesse; Frantz Fournier; Ivan Marc (pp. 1939-1949).

A new method to predict elementary amino acid (AA) composition of peptides (molar mass <1,000 g/mol) is described. This procedure is based on a computer-aided method using three combined analyses—reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and capillary electrophoresis coupled with mass spectrometry—and using a software calculating all possible amino acid combinations from the mass of any given peptide. The complementarity between HILIC and RPLC was demonstrated. Peptide retention prediction in HILIC was successfully modelled, and the achieved prediction accuracy was as high as r² = 0.97. This mathematical model, based on amino acid retention contributions and peptide length, provided the information about peptide hydrophilicity that was not redundant with its hydrophobicity. Correlations between respectively the hydrophobicity coefficients and RPLC retention time, hydrophilicity and HILIC retention time, and electrophoretic mobility and migration time were used for ranking all potential AA combinations corresponding to the given mass. The essential contribution of HILIC in this identification strategy and the need to combine the three models to significantly increase identification capabilities were both shown. Applied to an 18-standard peptide mixture, the identification procedure enabled the actual AA combination determination of the 14 di- to pentapeptides, in addition to an over 98 % reduction of possible combination numbers for the four hexapeptides. This procedure was then applied to the identification of 24 unknown peptides in a rapeseed protein hydrolysate. The effective AA composition was found for ten peptides, whereas for the 14 other peptides, the number of possible combinations was reduced by over 95 % thanks to the association of the three analyses. Finally, as a result of the information provided by the analytical techniques about peptides present in the mixture, the proposed method could become a highly valuable tool to recover bioactive peptides from undefined protein hydrolysates.

Keywords: HILIC-MS; Identification strategy; Peptides; Prediction method

Orthogonal array design for optimization of hollow-fiber-based liquid-phase microextraction combined with high-performance liquid chromatography for study of the pharmacokinetics of magnoflorine in rat plasma by Jun Zhou; Jiang Bing Sun; Ping Zheng; Jing Liu; Zhao Hui Cheng; Ping Zeng; Feng Qiao Wang (pp. 1951-1960).

In this work, a new sample-preparation method based on hollow-fiber liquid-phase microextraction (HF-LPME) was developed for analysis of magnoflorine in rat plasma. Analysis was accomplished by reversed-phase high-performance liquid chromatography (HPLC), with ultraviolet detection by use of a photodiode-array detector. An orthogonal array design (OAD) was found to be effective for optimization of major conditions which may affect the efficiency of HF-LPME. Under the optimized conditions (pH of donor and acceptor phases 12 and 2.0, respectively; extraction time 20 min; stirring speed 800 rpm; and addition of 10 % (w/v) salt), the preconcentration factor for magnoflorine was 355. Calibration curves with reasonable linearity (r ² ≥ 0.9994) were obtained in the range 10–1000 ng mL⁻¹. Intra-day and inter-day precision (RSD) were <5.5 % and the limit of detection (LOD) for the analyte was 3.0 ng mL⁻¹ (S/N = 3). The validated method was successfully used for pharmacokinetic studies of magnoflorine in rat plasma after intravenous administration.

Keywords: Magnoflorine; Hollow-fiber liquid-phase microextraction; Orthogonal array design; HPLC

Characteristics of the spin-trapping reaction of a free radical derived from AAPH: further development of the ORAC-ESR assay by A. Nakajima; E. Matsuda; Y. Masuda; H. Sameshima; T. Ikenoue (pp. 1961-1970).

The characteristics of the spin-trapping reaction in the oxygen radical absorbance capacity (ORAC)-electron spin resonance (ESR) assay were examined, focusing on the kind of spin traps. 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH) was used as a free radical initiator. The spin adducts of the AAPH-derived free radical were assigned as those of the alkoxyl radical, RO· (R = H₂N(HN)C–C(CH₃)₂). Among the spin traps tested, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5,5-dimethyl-4-phenyl-1-pyrroline N-oxide (4PDMPO), 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were applicable to the ORAC-ESR assay. Optimal formation of spin-trapped radical adduct was observed with 1 mM AAPH, 10 mM spin trap, and 5 s UV irradiation. The calibration curve (the Stern–Volmer’s plot) for each spin trap showed good linearity, and their slopes, k _SB/k _ST, were estimated to be 87.7 ± 2.3, 267 ± 15, 228 ± 9, and 213 ± 16 for DMPO, 4PDMPO, CYPMPO, and DEPMPO, respectively. Though the k _SB/k _ST values for selected biosubstances varied with various spin traps, their ratios to Trolox (the relative ORAC values) were almost the same for all spin traps tested. The ORAC-ESR assay also had a very good reproducibility. The ORAC-ESR assay was conducted under stoichiometric experimental conditions. The present results demonstrate the superiority of the ORAC-ESR assay. Structures of spin traps, ESR spectra of their spin adducts of AAPH-derived free radical, and their Stern-Volmer’s plots for Trolox.

Keywords: ORAC-ESR assay; AAPH; Spin-trapping; CYPMPO; DMPO

Colorimetric recognition and sensing of thiocyanate with a gold nanoparticle probe and its application to the determination of thiocyanate in human urine samples by Jia Zhang; Cheng Yang; Xiaolei Wang; Xiurong Yang (pp. 1971-1981).

A colorimetric method for the determination of thiocyanate (SCN⁻) ion with a cystamine-modified gold nanoparticle (Au NP) probe is presented. In this method, recognition is based on electrostatic attraction and directional hydrogen bonding between thiocyanate and cystamine on the surface of an Au NP. In phosphate buffer solution (PBS, pH 5.2, 10 mM), the cystamine-modified Au NPs readily aggregated upon incubation with N,N-dimethyl-1-naphthylamine (denoted “2N”), and a visible change in the color of the solution from red to blue was observed. When present, thiocyanate interacted with the gold nanoparticle probe more prominently than 2N, thereby protecting the gold nanoparticles and attenuating the degree of aggregation. The solution was observed (by the naked eye) to change in color from blue to purple and then back to red as a function of thiocyanate concentration (<10 μM). Iodide was noted to be a significant interferent; however, the optical absorption spectrum in the presence of iodide was fortunately easily distinguished from that for thiocyanate, thereby making it possible to discriminate iodide from thiocyanate. It was possible to determine thiocyanate in human urine samples using this method. This colorimetric method opens up a new avenue for assaying thiocyanate considering its rapid readout and simple implementation, which makes it convenient to determine thiocyanate in biological samples, especially at levels below 100 μM.

Keywords: Colorimetric; Gold nanoparticle; Thiocyanate; Anion; Sensor; Urine

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part I: screening strategy and pre-validation study with lipophilic marine toxins by Anne-Laure Sérandour; Aurélie Ledreux; Bénédicte Morin; Sylvain Derick; Elie Augier; Rachelle Lanceleur; Sahima Hamlaoui; Serge Moukha; Christophe Furger; Ronel Biré; Sophie Krys; Valérie Fessard; Marc Troussellier; Cécile Bernard (pp. 1983-1993).

Human poisoning due to consumption of seafood contaminated with phycotoxins is a worldwide problem, and routine monitoring programs have been implemented in various countries to protect human consumers. Following successive episodes of unexplained shellfish toxicity since 2005 in the Arcachon Bay on the French Atlantic coast, a national research program was set up to investigate these atypical toxic events. Part of this program was devoted to fit-for-purpose cell-based assays (CBA) as complementary tools to collect toxicity data on atypical positive-mouse bioassay shellfish extracts. A collaborative study involving five laboratories was conducted. The responses of human hepatic (HepG2), human intestinal (Caco2), and mouse neuronal (Neuro2a) cell lines exposed to three known lipophilic phycotoxins—okadaic acid (OA), azaspiracid-1 (AZA1), and pectenotoxin-2 (PTX2)—were investigated. A screening strategy composed of standard operating procedures and a decision tree for dose–response modeling and assay validation were designed after a round of “trial-and-error” process. For each toxin, the shape of the concentration–response curves and the IC₅₀ values were determined on the three cell lines. Whereas OA induced a similar response irrespective of the cell line (complete sigmoid), PTX2 was shown to be less toxic. AZA1 induced cytotoxicity only on HepG2 and Neuro2a, but not on Caco2. Intra- and inter-laboratory coefficients of variation of cell responses were large, with mean values ranging from 35 to 54 % and from 37 to 48 %, respectively. Investigating the responses of the selected cell lines to well-known toxins is the first step supporting the use of CBA among the panel of methods for characterizing atypical shellfish toxicity. Considering these successful results, the CBA strategy will be further applied to extracts of negative, spiked, and naturally contaminated shellfish tissues.

Keywords: Cell-based assays; Collaborative study; Lipophilic phycotoxins; Cytotoxicity

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part II: application to shellfish extracts spiked with lipophilic marine toxins by Aurélie Ledreux; Anne-Laure Sérandour; Bénédicte Morin; Sylvain Derick; Rachelle Lanceleur; Sahima Hamlaoui; Christophe Furger; Ronel Biré; Sophie Krys; Valérie Fessard; Marc Troussellier; Cécile Bernard (pp. 1995-2007).

Successive unexplained shellfish toxicity events have been observed in Arcachon Bay (Atlantic coast, France) since 2005. The positive mouse bioassay (MBA) revealing atypical toxicity did not match the phytoplankton observations or the liquid chromatography−tandem mass spectrometry (LC−MS/MS) investigations used to detect some known lipophilic toxins in shellfish. The use of the three cell lines (Caco2, HepG2, and Neuro2a) allows detection of azaspiracid-1 (AZA1), okadaic acid (OA), or pectenotoxin-2 (PTX2). In this study, we proposed the cell-based assays (CBA) as complementary tools for collecting toxicity data about atypical positive MBA shellfish extracts and tracking their chromatographic fractionation in order to identify toxic compound(s). The present study was intended to investigate the responses of these cell lines to shellfish extracts, which were either control or spiked with AZA1, OA, or PTX2 used as positive controls. Digestive glands of control shellfish were extracted using the procedure of the standard MBA for lipophilic toxins and then tested for their cytotoxic effects in CBA. The same screening strategy previously used with pure lipophilic toxins was conducted for determining the intra- and inter-laboratory variabilities of the responses. Cytotoxicity was induced by control shellfish extracts whatever the cell line used and regardless of the geographical origin of the extracts. Even though the control shellfish extracts demonstrated some toxic effects on the selected cell lines, the extracts spiked with the selected lipophilic toxins were significantly more toxic than the control ones. This study is a crucial step for supporting that cell-based assays can contribute to the detection of the toxic compound(s) responsible for the atypical toxicity observed in Arcachon Bay, and which could also occur at other coastal areas.

Keywords: Cell-based bioassays; Collaborative study; Shellfish extracts; Cytotoxicity; Lipophilic phycotoxins

Detection of melamine in milk by surface-enhanced Raman spectroscopy coupled with magnetic and Raman-labeled nanoparticles by Nazife Nur Yazgan; İsmail Hakkı Boyacı; Ali Topcu; Uğur Tamer (pp. 2009-2017).

A new method based on surface-enhanced Raman spectroscopy (SERS) has been developed for sensitive and rapid detection of melamine. Spherical magnetic-core gold-shell nanoparticles (AuNPs) and rod-shaped gold nanoparticles (nanorods) labeled with a Raman-active compound were used to form a complex with the melamine molecules. 5,5′-Dithiobis(2-nitrobenzoic acid) was used as Raman-active compound because it is readily adsorbed by a gold nanoparticle surface forming a self-assembled monolayer (SAM) and has strong Raman scattering at 1330 cm⁻¹, because of the symmetric NO₂ stretch. The calibration curve was obtained by plotting Raman band area at 1330 cm⁻¹ against melamine concentration. A linear relationship was obtained with a high determination coefficient (R ² = 0.997). The method was validated for linearity, sensitivity, precision (intra-day and inter-day repeatability), and recovery. In the model system, the limits of detection (LOD) and quantification (LOQ) were 0.38 and 1.27 mg L⁻¹, respectively. For melamine-spiked milk samples, LOD and LOQ values were 0.39 mg L⁻¹ and 1.30 mg L⁻¹, respectively. Intra and inter-day precision were 3.73 and 4.94 %, respectively. This method was applied to samples of skimmed milk that had been spiked with melamine at different concentrations. The recovery of the method was 95–109 % in the concentration range 2–15 mg L⁻¹, and average RSD was 1.71 %. Total analysis time was less than 15 min. Figure 1 Melamine acts as a cross linker molecule between the magnetic AuNPs and Raman-labeled nanorods

Keywords: Melamine; Surface-enhanced Raman spectroscopy; Magnetic nanoparticles; Milk

Development and validation of a direct competitive monoclonal antibody-based immunoassay for the sensitive and selective analysis of the phytoregulator forchlorfenuron by Celia Suárez-Pantaleón; Francesc A. Esteve-Turrillas; Josep V. Mercader; Consuelo Agulló; Antonio Abad-Somovilla; Antonio Abad-Fuentes (pp. 2019-2026).

Forchlorfenuron is a synthetic phytohormone with cytokinin-like activity used worldwide as a plant growth regulator to increase fruit size in a number of crops, mostly in kiwifruit and grape vines. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the determination of forchlorfenuron has been characterized and optimized. The selected immunoreagents afforded a highly selective assay with a limit of detection of 10 ng L⁻¹ in buffer. This direct competitive ELISA was validated in terms of trueness, precision, and robustness using both commercial juice and whole fruit samples. Recoveries from fortified kiwifruit juices and white and red musts were between 97 % and 131 %, with relative standard deviations below 16 %. When homogenized whole fruits were analysed after acetonitrile extraction, recoveries between 96 % and 113 % were found, with a limit of quantification of 5 μg kg⁻¹. The proposed immunoassay was validated by comparison with a reference chromatographic method using fruits from in-field treated grape and kiwifruit vines. Linear regression analysis of ELISA and HPLC–UV determinations showed an excellent correlation (r ² = 0.998), whereas analysis of the slope (0.99 ± 0.01) and of the intercept (−1 ± 3) clearly proved that the developed competitive immunoassay provided results that were statistically comparable to those obtained by the instrumental method for the analysis of forchlorfenuron in fruits at trace levels. Figure Monoclonal ELISA validation for forchlorfenuron

Keywords: CPPU; Cytokinin; Hapten; ELISA; Food safety; HPLC

Objective chemical fingerprinting of oil spills by partial least-squares discriminant analysis by M. P. Gómez-Carracedo; J. Ferré; J. M. Andrade; R. Fernández-Varela; R. Boqué (pp. 2027-2037).

An objective method based on partial least-squares discriminant analysis (PLS-DA) was used to assign an oil lump collected on the coastline to a suspected source. The approach is an add-on to current US and European oil fingerprinting standard procedures that are based on lengthy and rather subjective visual comparison of chromatograms. The procedure required an initial variable selection step using the selectivity ratio index (SRI) followed by a PLS-DA model. From the model, a “matching decision diagram” was established that yielded the four possible decisions that may arise from standard procedures (i.e., match, non-match, probable match, and inconclusive). The decision diagram included two limits, one derived from the Q-residuals of the samples of the target class and the other derived from the predicted y of the PLS model. The method was used classify 45 oil lumps collected on the Galician coast after the Prestige wreckage. The results compared satisfactorily with those from the standard methods.

Keywords: Oil spills; Discriminant PLS; Variable selection; Hydrocarbons

Sampling of tar from sewage sludge gasification using solid phase adsorption by Isabel Ortiz González; Rosa Ma Pérez Pastor; José Ma Sánchez Hervás (pp. 2039-2046).

Sewage sludge is a residue from wastewater treatment plants which is considered to be harmful to the environment and all living organisms. Gasification technology is a potential source of renewable energy that converts the sewage sludge into gases that can be used to generate energy or as raw material in chemical synthesis processes. But tar produced during gasification is one of the problems for the implementation of the gasification technology. Tar can condense on pipes and filters and may cause blockage and corrosion in the engines and turbines. Consequently, to minimize tar content in syngas, the ability to quantify tar levels in process streams is essential. The aim of this work was to develop an accurate tar sampling and analysis methodology using solid phase adsorption (SPA) in order to apply it to tar sampling from sewage sludge gasification gases. Four types of commercial SPA cartridges have been tested to determine the most suitable one for the sampling of individual tar compounds in such streams. Afterwards, the capacity, breakthrough volume and sample stability of the Supelclean™ ENVI-Carb/NH₂, which is identified as the most suitable, have been determined. Basically, no significant influences from water, H₂S or NH₃ were detected. The cartridge was used in sampling real samples, and comparable results were obtained with the present and traditional methods.

Keywords: Tar sampling; Gasification; Solid phase adsorption; Gas chromatography–mass spectrometry

Characterization of AhR agonist compounds in roadside snow by Martine Muusse; Katherine Langford; Knut Erik Tollefsen; Gerard Cornelissen; Peter Haglund; Ketil Hylland; Kevin V. Thomas (pp. 2047-2056).

Aryl hydrocarbon receptor (AhR) agonistic contaminants were identified in roadside snow samples. Snow was collected in Oslo, Norway, and compared to a background sample collected from a mountain area. The water and particulate fractions were analysed for AhR agonists using a dioxin-responsive, chemically activated luciferase expression (CALUX) cell assay and by gas chromatography coupled to high-resolution time-of-flight mass spectrometry with targeted analysis for polycyclic aromatic hydrocarbons (PAHs) and broad-spectrum non-target analysis. The AhR agonist levels in the dissolved fractions in the roadside samples were between 15 and 387 pg/L CALUX toxic equivalents (TEQ_CALUX). An elevated AhR activity of 221 pg TEQ_CALUX per litre was detected in the mountain sample. In the particle-bound fractions, the TEQ_CALUX was between 1,350 and 7,390 pg/L. One possible explanation for the elevated levels in the dissolved fraction of the mountain sample could be the presence of black carbon in the roadside samples, potentially adsorbing dioxin-like compounds and rendering them unavailable for AhR interaction. No polychlorinated dibenzodioxins and dibenzofurans or polychlorinated biphenyls were detected in the samples; the occurrence of PAHs, however, explained up to 9 % of the AhR agonist activity in the samples, whilst comprehensive two-dimensional gas chromatography coupled to mass spectrometry GCxGC-ToF-Ms identified PAH derivatives such as polycyclic aromatic ketones and alkylated, nitrogen sulphur and oxygen PAHs in the particle fractions. The (large) discrepancy between the total and explained activity highlights the fact that there are other as yet unidentified AhR agonists present in the environment. Figure

Keywords: Snow; Effect-directed analysis; CALUX; Black carbon

2-Nitro-6-monoacetylmorphine: potential marker for monitoring the presence of 6-monoacetylmorphine in urine adulterated with potassium nitrite by Susan Luong; Ronald Shimmon; James Hook; Shanlin Fu (pp. 2057-2063).

6-Monoacetylmorphine (6-MAM), being a unique metabolite of heroin, is routinely tested in urine samples to monitor heroin use. However, detection of 6-MAM-related opiates such as morphine is known to be affected by in vitro urine adulteration using oxidizing adulterants such as potassium nitrite. This study aimed to investigate the fate of 6-MAM after exposure to nitrite and to identify any formed oxidation products that may potentially be used for monitoring heroin abuse despite nitrite adulteration. Potassium nitrite (0.05 M and 0.6 M) was reacted with 6-MAM (5–10,000 ng/mL) in both water and blank urine with pH adjusted to range from 3 to 8. Following reaction at room temperature for varying periods, the reaction mixtures were monitored by both the CEDIA® Heroin Metabolite (6-AM) immunoassay and liquid chromatography-mass spectrometry (LC-MS) methods. Structural elucidation of the isolated oxidation products was based on mass spectrometry and nuclear magnetic resonance spectroscopic evidence. Nitrite, under acidic environment (pH < 7), was shown to be effective in masking the detection of 6-MAM by both the CEDIA® immunoassay and the LC-MS methods. 2-Nitro-6-monoacetylmorphine (2-nitro-MAM) was identified as the sole oxidation product, which remained detectable in urine for at least 11 days under the experimental conditions investigated. 2-Nitro-MAM was detectable in a urine sample of a heroin user after nitrite exposure. 2-Nitro-MAM has shown potential to serve as a marker for monitoring heroin abuse when urine is adulterated with nitrite. Certification of 2-nitro-MAM reference standard for further development of its quantitative testing methods is thus warranted.

Keywords: 6-Monoacetylmorphine (6-MAM); 2-Nitro-6-monoacetylomorphine (2-nitro-MAM); Heroin; Urine adulteration; Nitrite; Oxidation

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Analytical and Bioanalytical Chemistry (v.403, #7)