Determination of dihydroartemisinic acid in Artemisia annua L. by gas chromatography with flame ionization detection

Dihydroartemisinic acid (DHAA) is the direct precursor to artemisinin, an effective anti-malaria compound from Artemisia annua L. (A. annua), and it can be transformed to artemisinin without the catalysis of enzyme. A rapid and sensitive analysis of DHAA in A. annua is needed to screen excellent plant resources aimed to improve artemisinin production. In order to develop a rapid and sensitive determination method for DHAA in plant, the extraction and analysis conditions were extensively investigated in the present work. As a result, extraction of powdered A. annua leaves at 55 °C for 50 min with chloroform resulted in the highest yield of DHAA, with a recovery of more than 98%. The precision of this gas chromatographic procedure ranged from 1.22 to 2.94% for intra-day and from 1.69 to 4.31% for inter-day, respectively. The accuracy was 99.55-103.02% for intra-day and 98.86-99.98 % for inter-day, respectively. The measured LOQ and LOD values of the proposed method reached 5.00 and 2.00 µg/mL, respectively. Validation indicated the method was robust, quick, sensitive and adequate for DHAA analysis.



A fast and sensitive UHPLC-MS/MS method for the determination of N-butylscopolamine in human plasma: application in a bioequivalence study

We have developed and validated a fast and sensitive ultra high-performance liquid chromatography with positive ion electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for determining N-butylscopolamine levels in human plasma using propranolol as an internal standard (IS). The acquisition was set up in the multiple reaction monitoring (MRM) mode with the transitions m/z 360.2  138.0 for N-butylscopolamine and m/z 260.2  116.1 for IS. This method uses a liquid-liquid extraction process with dichloromethane. The analyte and IS were chromatographed on a C18 – 50 x 2.1 mm, 1.7-µm column through isocratic elution with acetonitrile:5-mM ammonium acetate (adjusted to pH 3.0 with formic acid). The method was linear in the 1 to 1000 pg/mL range for N-butylscopolamine and was selective, precise, accurate, and robust. The validated method was successfully applied to perform a bioequivalence study of the reference (Buscopan®, from Boehringer Ingelheim) and the test sample coated-tablet formulations (from Foundation for Popular Remedy [FURP]), both containing 10 mg of N-butylscopolamine bromide administered as a single dose. Using 58 healthy volunteers and accounting for the high intra-individual variability confirmed by statistical calculations (38%), the two formulations were considered bioequivalent because the rate and extent of absorption (within 80–125% interval), satisfying international requirements.



Numerical Simulation and Experimental Validation of Calibrant-Loaded Extraction Phase Standardization Approach

Analytical ChemistryDOI: 10.1021/acs.analchem.6b01802



Colorimetric Sensor Array for Thiols Discrimination Based on Urease–Metal Ion Pairs

Analytical ChemistryDOI: 10.1021/acs.analchem.6b01493



Polytetrafluoroethylene-jacketed stirrer modified with graphene oxide and polydopamine for the efficient extraction of polycyclic aromatic hydrocarbons

Steel stirrers jacketed with polytetrafluoroethylene can be regarded as an ideal substrate for stirrer bar sorptive extraction. However, it is still a great challenge to immobilize graphene onto a polytetrafluoroethylene stirrer due to the highly chem…



Development and validation of a LC-MS/MS quantitative method for endogenous deoxynucleoside triphosphates in cellular lysate

The endogenous deoxynucleoside triphosphate (dNTP) pool includes deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and thymidine triphosphate (TTP). The endogenous dNTP pool is regulated by complex enzymatic pathways that can be targeted by drugs, such as antimetabolites. In addition, these components compete with antiviral nucleos(t)ide analog triphosphates, contributing to the mechanism of pharmacologic action. This communication describes the development and validation of a sensitive method to quantify the intracellular dNTP pool in cellular lysates. The extraction process was optimized for dNTPs using an indirect strategy—the separation of mono-, di-, and triphosphate moieties by strong anion exchange, dephosphorylation of target fractions to molar equivalent nucleosides—followed by sensitive quantitation using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The validated analytical range was 50 to 2500 fmol/sample for each dNTP. The assay was used to quantify dNTPs in peripheral blood mononuclear cells (PBMC) from forty clinical research participants (n = 279 samples). Median (interquartile range) concentrations were 143 (116, 169) for dATP, 737 (605, 887) for dCTP, 237 (200, 290) for dGTP, and 315 (220, 456) for TTP, respectively, in femtomole per million cells. This method allows for future studies of endogenous dNTP disposition in subjects receiving antimetabolites, nucleos(t)ide analogues, or for other clinical scenarios.



A New Validated HPLC Method for the Determination of Quercetin: Application to Study Pharmacokinetics in Rats

A simple, accurate, and reproducible High Performance Liquid Chromatography (HPLC) method has been developed and validated for the quantification of quercetin (QR) in rat plasma. The method involves a simple protein precipitation procedure to extract both QR and thymoquinone (TQ), the internal standard. The chromatographic analysis was achieved on a Shimadzu LC 20A HPLC system equipped with a Supelcosil LC-18 T C18 column and an isocratic mobile phase consisted of 0.3% trichloroacetic acid in water and acetonitrile HPLC grade (50:50, v/v) run at a flow rate of 0.9 ml/min for 13 minutes. The UV detection wavelength was set at 254 nm. The method exhibited good linearity (R2 > 0.994) over the assayed concentration range (0.10-25 µg/ml) and demonstrated good intra-day and inter-day precision and accuracy (relative standard deviations and the deviation from predicted values were less than 20%). This method was also successfully applied for studying the pharmacokinetics of QR in rats following a single oral dose of QR to evaluate its pharmacokinetic parameters in rats.



Determination of MLN0128, an investigational antineoplastic agent, in human plasma by LC-MS/MS.

MLN0128, an mTOR kinase inhibitor, is currently undergoing clinical investigation for treatment of a variety of cancers. To support this work, an LC-MS/MS method has been developed for determination of MLN0128 in human plasma. A structural analog STK040263 was used as the internal standard. Both MLN0128 and the IS were first extracted from plasma using methyl tert-butyl ether; then separated on a Waters XTerra® MS C18 column using a mobile phase consisting of methanol/acetonitrile/10.0 mM ammonium formate (34:6:60, v/v/v) at a flow rate of 0.300 ml min-1. Quantitation of MLN0128 was done by positive electrospray ionization tandem mass spectrometry in multiple-reaction-monitoring mode. This method has a total run time of <4 min with the retention times of 1.95 min and 2.94 min for the IS and MLN0128, respectively. The method has been validated per the US-FDA guidance for bioanalytical method validation. It has a calibration range of 0.100-50.0 ng mL-1 in human plasma with a correlation coefficient >0.999. The overall assay accuracy and precision were ≤ ±4% and ≤8%, respectively. The IS normalized recovery of MLN0128 ranged 98-100%. The stability studies showed that MLN0128 was stable under all tested conditions. The method developed may be useful for clinical studies of MLN0128.



Chemiluminescence imaging for microRNA detection based on cascade exponential isothermal amplification machinery

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Yongjie Xu, Dandan Li, Wei Cheng, Rong Hu, Ye Sang, Yibing Yin, Shijia Ding, Huangxian Ju
A novel G-quadruplex DNAzyme-driven chemiluminescence (CL) imaging method was developed for ultrasensitive and specific detection of miRNA based on the cascade exponential isothermal amplification reaction (EXPAR) machinery. A structurally tailored hairpin probe switch was designed to selectively recognise miRNA and form hybridisation products to trigger polymerase and nicking enzyme machinery, resulting in the generation of product I, which was complementary to a region of the functional linear template. Then, the response of the functional linear template to the generated product I further activated the exponential isothermal amplification machinery, leading to synthesis of numerous horseradish peroxidase mimicking DNAzyme units for CL signal transduction. The amplification paradigm generated a linear response from 10 fM to 100 pM, with a low detection limit of 2.91 fM, and enabled discrimination of target miRNA from a single-base mismatched target. The developed biosensing platform demonstrated the advantages of isothermal, homogeneous, visual detection for miRNA assays, offering a promising tool for clinical diagnosis.

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Aptamer-based biosensor for label-free detection of ethanolamine by electrochemical impedance spectroscopy

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Gang Liang, Yan Man, Xinxin Jin, Ligang Pan, Xinhui Liu
A label-free sensing assay for ethanolamine (EA) detection based on G-quadruplex-EA binding interaction is presented by using G-rich aptamer DNA (Ap-DNA) and electrochemical impedance spectroscopy (EIS). The presence of K+ induces the Ap-DNA to form a K+-stabilized G-quadruplex structure which provides binding sites for EA. The sensing mechanism was further confirmed by circular dichroism (CD) spectroscopy and EIS measurement. As a result, the charge transfer resistance (RCT) is strongly increased as demonstrated by using the ferro/ferricyanide ([Fe(CN)6]3−/4−) as a redox probe. Under the optimized conditions, a linear relationship between ΔRCT and EA concentration was obtained over the range of 0.16 nM and 16 nM EA, with a detection limit of 0.08 nM. Interference by other selected chemicals with similar structure was negligible. Analytical results of EA spiked into tap water and serum by the sensor suggested the assay could be successfully applied to real sample analysis. With the advantages of high sensitivity, selectivity and simple sensor construction, this method is potentially suitable for the on-site monitoring of EA contamination.

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Click-modified hexahomotrioxacalix[3]arenes as fluorometric and colorimetric dual-modal chemosensors for 2,4,6-trinitrophenol

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Chong Wu, Jiang-Lin Zhao, Xue-Kai Jiang, Xin-Long Ni, Xi Zeng, Carl Redshaw, Takehiko Yamato
A new type of chemosensor-based approach to the detection of 2,4,6-trinitrophenol (TNP) is described in this paper. Two hexahomotrioxacalix[3]arene-based chemosensors 1 and 2 were synthesized through click chemistry, which exhibited high binding affinity and selectivity toward TNP as evidenced by UV–vis and fluorescence spectroscopy studies. 1H NMR titration analysis verified that CH⋯O hydrogen bonding is demonstrated as the mode of interaction, which possibly facilitates effective charge-transfer.

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DNA aptamers for selective identification and separation of flame retardant chemicals

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Un-Jung Kim, Byoung Chan Kim
Polybrominated diphenyl ethers (PBDEs) are group of chemicals which are representative persistent organic pollutants (POPs) and used as brominated flame retardants for many consumer products. PBDEs were phased out since 2009 but are still frequently observed in various environmental matrices and human body. Here, we report ssDNA aptamers which bind to BDE47, one of the PBDE congeners commonly found in various environmental matrices, and show affinity to other major tri-to hepta- BDE congeners. The PBDE specific aptamers were isolated from random library of ssDNA using Mag-SELEX. Two out of 15 sequences, based on their alignment and hairpin loop structures, were chosen to determine dissociation constant with BDE47 and showed from picomolar to nanomolar affinities (200 pM and 1.53 nM). The aptamers displayed high selectivity to the original target, BDE47, and implying general specificity to PBDE backbone with varying affinities to other congeners. Further, we showed that the use of two aptamers together could enhance the separation efficiency of BDE47 and other BDE congeners when dissolved in a solvent compared to use of single aptamer. These aptamers are expected to provide a tool for preliminary screening or quick separation of PBDEs in environmental samples prior to trace quantitative analysis.

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A novel detection of radon based on its decay product inducing conformational changes of an aptamer probe

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Minzhi Long, Han Deng, Gang Tian, Chunli Song, Hongwen Liu, Yi Shen, Changyin Lv
This study proposes a novel method for the detection of inert gas radon using a label-free, specific, fluorescence-sensing aptamer in the context of PW17-OG system. This method utilizes the cyanine dye OliGreen (OG) as a signal reactor and the aptamer PW17 as a fluorescent identification probe. When OG integrates into the free curling PW17, a strong fluorescence signal is generated. After radon decays, the long lived naturally occurring radon progeny Pb being disposed and introduced to the system. Lead ions induce PW17 to form a stable G-quadruplex, thereby inhibiting the interaction between OG and PW17 and resulting in a reduction of the fluorescence intensity. The fluorescence intensity show a good linear relationship with lead ion and the radon concentration (D), thereinto, We fitted linear regression of radon concentration in the range of 0.92–4.22 (×104 Bqhm−3) to receive a good relationship between ΔF and the concentration of radon with the detection limit of 1963 Bqhm−3. This method has been successfully applied for detecting standard cumulative concentration of radon and the detection limit reached the national standard of China. This sensitive method can exclude radiation damage in field testing, furthermore, it explores a new field in biological analysis using an aptamer to detected inorganic, gaseous, and radioactive materials.

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Fluorescence ELISA for sensitive detection of ochratoxin A based on glucose oxidase-mediated fluorescence quenching of CdTe QDs

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Yi Liang, Xiaolin Huang, Ruijin Yu, Yaofeng Zhou, Yonghua Xiong
The present study described a novel fluorescence enzyme-linked immunosorbent assay (ELISA) used to detect ochratoxin A (OTA) by using the glucose oxidase (GOx)-mediated fluorescence quenching of mercaptopropionic acid-capped CdTe quantum dots (MPA-QDs), in which GOx was used as an alternative to horseradish peroxidase (HRP) for the oxidization of glucose into hydrogen peroxide (H2O2) and gluconic acid. The MPA-QDs were used as a fluorescent signal output, whose fluorescence variation was extremely sensitive to the presence of H2O2 or hydrogen ions in the solution. Under the optimized conditions, the proposed fluorescence ELISA demonstrated a good linear detection of OTA in corn extract from 2.4 pg mL−1 to 625 pg mL−1 with a limit of detection of 2.2 pg mL−1, which was approximately 15-fold lower than that of conventional HRP-based ELISA. Our developed fluorescence immunoassay was also similar to HRP-based ELISA in terms of selectivity, accuracy, and reproducibility. In summary, this study was the first to use the GOx-mediated fluorescence quenching of QDs in immunoassay to detect OTA, offering a new possibility for the analysis of other mycotoxins and biomolecules.

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Formation of core-shell Au@Ag nanorods induced by catecholamines: A comparative study and an analytical application

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): M.V. Gorbunova, V.V. Apyari, S.G. Dmitrienko, A.V. Garshev
Gold nanorods (AuNRs) stabilized by cetyltrimethylammonium bromide (CTAB) were synthesized and an interaction of catecholamines (CAs) with silver ions in the presence of the obtained AuNRs was studied. The reaction results into formation of core-shell Au@Ag nanorods (Au@AgNRs) and leads to a hypsochromic shift of the long-wave surface plasmon resonance (SPR) band in the absorption spectrum of AuNRs. The influence of a CA structure, excess of CTAB, interaction time, pH, concentration of AuNRs, silver ions and CAs on this interaction was studied. Based on correlation of the NRs spectral characteristics with the concentration of CAs, a method for spectrophotometric determination of dobutamine, epinephrine, norepinephrine and dopamine with detection limits 27, 18, 16 and 13 μg L−1, respectively, has been developed. The method can be applied to the analysis of medicines.

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Magnetic deep eutectic solvents molecularly imprinted polymers for the selective recognition and separation of protein

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Yanjin Liu, Yuzhi Wang, Qingzhou Dai, Yigang Zhou
A novel and facile magnetic deep eutectic solvents (DES) molecularly imprinted polymers (MIPs) for the selective recognition and separation of Bovine hemoglobin (BHb) was prepared. The new-type DES was adopted as the functional monomer which would bring molecular imprinted technology to a new direction. The amounts of DES were optimized. The obtained magnetic DES-MIPs were characterized with fourier transform infrared spectrometry (FT-IR), thermogravimetric analysis (TGA), field emission scanning electron microscope (FESEM), dynamic light scattering (DLS), elemental analysis and vibrating sample magnetometer (VSM). The results suggested that the imprinted polymers were successfully formed and possessed a charming magnetism. The maximum adsorption capability (Qmax) and dissociation constant (KL) were analyzed by Langmuir isotherms (R2 = 0.9983) and the value were estimated to be 175.44 mg/g and 0.035 mg/mL for the imprinted particles. And the imprinted particles showed a high imprinting factor of 4.77. In addition, the magnetic DES-MIPs presented outstanding recognition specificity and selectivity so that it can be utilized to separate template protein from the mixture of proteins and real samples. Last but not least, the combination of deep eutectic solvents and molecular imprinted technology in this paper provides a new perspective for the recognition and separation of proteins.

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A simple sheathless CE-MS interface with a sub-micrometer electrical contact fracture for sensitive analysis of peptide and protein samples

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Tam T.T.N. Nguyen, Nickolaj J. Petersen, Kasper D. Rand
Online coupling of capillary electrophoresis (CE) to electrospray ionization mass spectrometry (MS) has shown considerable potential, however, technical challenges have limited its use. In this study, we have developed a simple and sensitive sheathless CE-MS interface based on the novel concept of forming a sub-micrometer fracture directly in the capillary. The simple interface design allowed the generation of a stable ESI spray capable of ionization at low nanoliter flow-rates (45–90 nL/min) for high sensitivity MS analysis of challenging samples like those containing proteins and peptides. By analysis of a model peptide (leucine enkephalin), a limit of detection (LOD) of 0.045 pmol/μL (corresponding to 67 attomol in a sample volume of ∼15 nL) was obtained. The merit of the CE-MS approach was demonstrated by analysis of bovine serum albumin (BSA) tryptic peptides. A well-resolved separation profile was achieved and comparable sequence coverage was obtained by the CE-MS method (73%) compared to a representative UPLC-MS method (77%). The CE-MS interface was subsequently used to analyse a more complex sample of pharmaceutically relevant human proteins including insulin, tissue factor and α-synuclein. Efficient separation and protein ESI mass spectra of adequate quality could be achieved using only a small amount of sample (30 fmol). In addition, analysis of ubiquitin samples under both native and denatured conditions, indicate that the CE-MS setup can facilitate native MS applications to probe the conformational properties of proteins. Thus, the described CE-MS setup should be useful for a wide range of high-sensitivity applications in protein research.

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A liquid chromatography – tandem mass spectrometry method to measure a selected panel of uremic retention solutes derived from endogenous and colonic microbial metabolism

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Henriette de Loor, Ruben Poesen, Wout De Leger, Wim Dehaen, Patrick Augustijns, Pieter Evenepoel, Björn Meijers
Chronic kidney disease (CKD) is associated with an increased risk of mortality and cardiovascular disease, which is, at least partly, mediated by the accumulation of so-called uremic retention solutes. Although there has been an increasing interest in the behavior of these solutes, derived from both the endogenous and colonic microbial metabolism, methods to simultaneously and accurately measure a broad panel of relevant uremic retention solutes remain scarce.We developed a highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. A high throughput sample preparation was used with extraction of analytes from 50 μl serum using Ostro plate technology. For most solutes, stable isotopes labelled metabolites were used as internal standards. Chromatography was achieved using an Acquity UPLC CSH Fluoro Phenyl column. The total run time was 8 min, the mobile phase was a gradient of 0.1% formic acid in Milli-Q water and pure methanol at a flow rate of 0.5 ml min−1. Detection was performed using a tandem mass spectrometer with alternated positive and negative electrospray ionization.Calibration curves were linear for all solutes. Precision was assessed according to the NCCLS EP5-T guideline, being below 15% for all metabolites. Mean recoveries were between 83 and 104% for all metabolites. The validated method was successfully applied in a cohort of 488 patients with CKD.We developed and validated a sensitive and robust UPLC-MS/MS method for quantification of 15 uremic retention solutes derived from endogenous and colonic microbial metabolism. This method allows for studying the behavior and relevance of these solutes in patients with CKD.

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UPLC-MS/MS detection of disaccharides derived from glycosaminoglycans as biomarkers of mucopolysaccharidoses

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Christiane Auray-Blais, Pamela Lavoie, Shunji Tomatsu, Vassili Valayannopoulos, John J. Mitchell, Julian Raiman, Maxime Beaudoin, Bruno Maranda, Joe T.R. Clarke
Mucopolysaccharidoses (MPSs) are a group of disorders resulting from primary defects in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Depending on the specific enzyme defect, the catabolism of one or more GAGs is blocked leading to accumulation in tissues and biological fluids. GAG measurements are important for high-risk screening, diagnosis, monitoring treatment efficacy, and patient follow up. The dimethylmethylene blue (DMB) spectrophotometric method commonly used in most biochemical genetics laboratories relies on a non-specific total GAG analysis which has led to false positive results, and even false negative results (mainly for MPS III and IV patients). The main objective of our project was to devise and validate a reliable tandem mass spectrometry multiplex analysis for the urine quantitation of four GAGs (dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)) for an eventual technological transfer to the clinic. The developed methodology is rapid (7 min) and our results showed good intraday and interday precision (RSDs ≤ 8.7%) and accuracy (Biases range: −12.0%–18.4%). Linearity was good (r2 &gt; 0.995) for DS, HS, CS, and KS calibration curves. In comparison with the DMB spectrophotometric method, this multiplex tandem mass spectrometry method allows GAG fractionation, thus a differentiation of MPS types, except for MPS I and II which are characterized by the same GAG profile. The devised method is a useful and reliable tool for diagnosis of MPS patients, as well as their monitoring and follow up, as shown by longitudinal studies.

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Screening halogenated environmental contaminants in biota based on isotopic pattern and mass defect provided by high resolution mass spectrometry profiling

Publication date: 14 September 2016
Source:Analytica Chimica Acta, Volume 936
Author(s): Ronan Cariou, Elsa Omer, Alexis Léon, Gaud Dervilly-Pinel, Bruno Le Bizec
In the present work, we addressed the question of global seeking/screening organohalogenated compounds in a large panel of complex biological matrices, with a particular focus on unknown chemicals that may be considered as potential emerging hazards. A fishing strategy was developed based on untargeted profiling among full scan acquisition datasets provided by high resolution mass spectrometry. Since large datasets arise from such profiling, filtering useful information stands as a central question. In this way, we took advantage of the exact mass differences between Cl and Br isotopes. Indeed, our workflow involved an innovative Visual Basic for Applications script aiming at pairing features according to this mass difference, in order to point out potential organohalogenated clusters, preceded by an automated peak picking step based on the centWave function (xcms package of open access R programming environment). Then, H/Cl-scale mass defect plots were used to visualize the datasets before and after filtering. The filtering script was successfully applied to a dataset generated upon liquid chromatography coupled to ESI(−)-HRMS measurement from one eel muscle extract, allowing for realistic manual investigations of filtered clusters. Starting from 9789 initial obtained features, 1994 features were paired in 589 clusters. Hexabromocyclododecane, chlorinated paraffin series and various other compounds have been identified or tentatively identified, allowing thus broad screening of organohalogenated compounds in this extract. Although realistic, manual review of paired clusters remains time consuming and much effort should be devoted to automation.

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