Analytical and Bioanalytical Chemistry (v.401, #2)
Anti-doping analysis by Mario Thevis (387-388).
is Professor at the German Sport University Cologne and Head of the Center for Preventive Doping Research since 2006. His research interests include the development of methods targeting non-approved emerging drugs with potential for misuse in sport, elucidation of metabolic pathways of drug candidates, and, particularly, the identification of peptidic drugs and their metabolites in blood and urine. He has published over 160 articles in peer-reviewed international journals, authored a book on mass spectrometry in sports drug testing, received the Manfred–Donike (2001) and Beynon–Price (2010) awards, and is Editor-in-Chief of Drug Testing and Analysis.
Surface plasmon resonance in doping analysis by R Gutiérrez-Gallego; E Llop; J Bosch; J Segura (389-403).
Doping analysis relies on the determination of prohibited substances that should not be present in the body of an athlete or that should be below a threshold value. In the case of xenobiotics their mere presence is sufficient to establish a doping offence. However, in the case of human biotics the analytical method faces the difficulty of distinguishing between endogenous and exogenous origin. For this purpose ingenious strategies have been implemented, often aided by state-of-the-art technological advancements such as mass spectrometry in all its possible forms. For larger molecules, i.e. protein hormones, the innate structural complexity, the heterogeneous nature, and the extremely low levels in biological fluids have rendered the analytical procedures heavily dependent of immunological approaches. Although approaches these confer specificity and sensitivity to the applications, most rely on the use of two, or even three, antibody incubations with the consequent increment in assay variability. Moreover, the requirement for different antibodies that separately recognise different epitopes in screening and confirmation assays further contributes to differences encountered in either measurement. The development of analytical techniques to measure interactions directly, such as atomic force microscopy, quartz crystal microbalance or surface plasmon resonance, have greatly contributed to the accurate evaluation of molecular interactions in all fields of biology, and expectations are that this will only increase. Here, an overview is provided of surface plasmon resonance, and its particular value in application to the field of doping analysis. Figure SPR analysis to assist antidoping procedures
Keywords: Surface plasmon resonance; Doping analysis; Proteins and immunoassay
Current role of LC-MS(/MS) in doping control by Mario Thevis; Andreas Thomas; Wilhelm Schänzer (405-420).
Liquid chromatography–(tandem) mass spectrometry [(LC-MS(/MS)] has become an integral part of modern sports drug testing as it offers unique capabilities complementing immunological and gas chromatography–(tandem) mass spectrometry [(GC-MS(/MS)]-based detection methods for prohibited compounds. The improved options of fast and sensitive targeted analysis as well as untargeted screening procedures utilizing high resolution/high accuracy mass spectrometry have considerably expanded the tools available to anti-doping laboratories for initial testing and confirmation methods. One approach is to focus on pre-selected target analytes that are measured with utmost specificity and sensitivity using diagnostic precursor–product ion pairs in low resolution tandem mass spectrometers. The other scenario is to measure and plot extracted ion chromatograms of protonated or deprotonated molecules as well as product ions as recorded in the full scan mode with high resolution/high accuracy mass spectrometry. Examples of recent applications of sports drug testing procedures published between 2007 and 2010 are presented and discussed, outlining the particular advantages of the selected approaches as well as their limitations in a short- and long-term perspective. Figure The utility of LC-MS(/MS) in current sports drug testing programs is discussed and trends in doping control methods as well as data mining are presented
Keywords: Doping control; Sport; Mass spectrometry; High resolution; Screening; Retrospective analysis
Prevalence of legal and illegal stimulating agents in sports by K. Deventer; K. Roels; F. T. Delbeke; P. Van Eenoo (421-432).
This paper reviews the prevalence of legal and illegal stimulants in relation to doping-control analysis. Stimulants are among the oldest classes of doping agents, having been used since ancient times. Despite the ease with which they can be detected and the availability of sensitive detection methods, stimulants are still popular among athletes. Indeed, they remain one of the top three most popular classes of prohibited substances. Because the list of legal and illegal stimulants is extensive only a selection is discussed in detail. The compounds selected are caffeine, ephedrines, amphetamine and related compounds, methylphenidate, cocaine, strychnine, modafinil, adrafinil, 4-methyl-2-hexaneamine, and sibutramine. These compounds are mainly prevalent in sport or are of therapeutic importance. Because stimulants are the oldest doping class the first detection methods were for this group. Several early detection techniques including GC–NPD, GC–ECD, and TLC are highlighted. The more novel detection techniques GC–MS and LC–MS are also discussed in detail. In particular, the last technique has been shown to enable successful detection of stimulants difficult to detect by GC–MS or for stimulants previously undetectable. Because stimulants are also regularly detected in nutritional (food) supplements a section on this topic is also included.
Keywords: Doping; Mass spectrometry; Amphetamine; Stimulants; Cocaine; Ephedrines
Recent developments in the use of isotope ratio mass spectrometry in sports drug testing by Thomas Piper; Caroline Emery; Martial Saugy (433-447).
According to the annual report of the World Anti-Doping Agency, steroids are the most frequently detected class of doping agents. Detecting the misuse of endogenously occurring steroids, i.e. steroids such as testosterone that are produced naturally by humans, is one of the most challenging issues in doping control analysis. The established thresholds for urinary concentrations or concentration ratios such as the testosterone/epitestosterone quotient are sometimes inconclusive owing to the large biological variation in these parameters.For more than 15 years, doping control laboratories focused on the carbon isotope ratios of endogenous steroids to distinguish between naturally elevated steroid profile parameters and illicit administration of steroids. A variety of different methods has been developed throughout the last decade and the number of different steroids under investigation by isotope ratio mass spectrometry has recently grown considerably. Besides norandrosterone, boldenone was found to occur endogenously in rare cases and the misuse of corticosteroids or epitestosterone can now be detected with the aid of carbon isotope ratios as well. In addition, steroids excreted as sulfoconjugates were investigated, and the first results regarding hydrogen isotope ratios recently became available.All of these will be presented in detail within this review together with some considerations on validation issues and on identification of parameters influencing steroidal isotope ratios in urine.
Keywords: Carbon isotope ratio; Hydrogen isotope ratio; Steroid profile; Steroid metabolism; Doping control
Detecting growth hormone abuse in athletes by Richard I. G. Holt (449-462).
It is believed that athletes have been abusing growth hormone (GH) for its anabolic and lipolytic effects since the early 1980s, at least a decade before endocrinologists began to treat adults with GH deficiency. There is an on-going debate about whether GH is performance enhancing. Although many of the early studies were negative, more recent studies suggest that GH improves strength and sprint capacity, particularly when it is combined with anabolic steroids. Although use of GH is banned by the World Anti-Doping Agency (WADA), its detection remains challenging. Two approaches have been developed to detect GH abuse. The first is based on measurement of pituitary GH isoforms; after injection of recombinant human GH, which comprises solely the 22-kDa isoform, endogenous production is down-regulated leading to an increase in the 22-kDa isoform relative to other isoforms. The second is based on measurement of markers of GH action. Insulin-like growth factor-I (IGF-I) and N-terminal pro-peptide of type III collagen (P-III-NP) increase in response to GH administration in a dose-dependent manner. When combined with discriminant function analysis, use of these markers differentiates between individuals taking GH and placebo. Subsequent studies have shown that the test is applicable across different ethnicities and is unaffected by injury. WADA regulations state that when analytes are measured by immunoassay, two assays are needed. Final validation of the marker test is currently being undertaken with modern commercially available immunoassays to finalise the threshold values to be used to determine whether a doping offence has been committed. Figure
Keywords: Growth hormone; Performance; Clinical trial; IGF-I; P-III-NP; Discriminant function
Recent developments in doping testing for erythropoietin by Christian Reichel (463-481).
The constant development of new erythropoiesis-stimulating agents (ESAs), since the first introduction of recombinant erythropoietin (rhEpo) for clinical use, has also necessitated constant development of methods for detecting the abuse of these substances. Doping with ESAs is prohibited according to the World Anti-Doping Code and its prohibited list of substances and methods. Since the first publication of a direct and urine-based detection method in 2000, which uses changes in the Epo isoform profile as detected by isoelectric focusing in polyacrylamide slab gels (IEF-PAGE), the method has been constantly adapted to the appearance of new ESAs (e.g., Dynepo, Mircera). Blood had to be introduced as an additional matrix, because Mircera (a PEGylated Epo) is best confirmed in serum or plasma after immunoaffinity purification. A Mircera ELISA was developed for fast screening of sera. With the appearance of Dynepo and copy epoetins, the additional application of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE or equivalent) became necessary. The haematological module of the Athlete Biological Passport is the latest development in multivariable indirect testing for ESA doping. The article summarizes the main strategies currently used in Epo anti-doping testing with special focus on new developments made between 2009 and 2010. Figure Recent developments in doping testing for Epo include mass-based methods. The apparent molecular mass of most rhEpos is higher than for uhEpo, which can be shown by SDS-PAGE
Keywords: Erythropoietin (Epo); Doping control; Electrophoresis; Mass spectrometry; ELISA; Biological passport; Direct and indirect detection
The importance of reference materials in doping-control analysis by Lindsey G. Mackay; Rymantas Kazlauskas (483-492).
Currently a large range of pure substance reference materials are available for calibration of doping-control methods. These materials enable traceability to the International System of Units (SI) for the results generated by World Anti-Doping Agency (WADA)-accredited laboratories. Only a small number of prohibited substances have threshold limits for which quantification is highly important. For these analytes only the highest quality reference materials that are available should be used. Many prohibited substances have no threshold limits and reference materials provide essential identity confirmation. For these reference materials the correct identity is critical and the methods used to assess identity in these cases should be critically evaluated. There is still a lack of certified matrix reference materials to support many aspects of doping analysis. However, in key areas a range of urine matrix materials have been produced for substances with threshold limits, for example 19-norandrosterone and testosterone/epitestosterone (T/E) ratio. These matrix-certified reference materials (CRMs) are an excellent independent means of checking method recovery and bias and will typically be used in method validation and then regularly as quality-control checks. They can be particularly important in the analysis of samples close to threshold limits, in which measurement accuracy becomes critical. Some reference materials for isotope ratio mass spectrometry (IRMS) analysis are available and a matrix material certified for steroid delta values is currently under production. In other new areas, for example the Athlete Biological Passport, peptide hormone testing, designer steroids, and gene doping, reference material needs still need to be thoroughly assessed and prioritised.
Keywords: Certified reference materials; Reference materials; Doping
Structure characterisation of urinary metabolites of the cannabimimetic JWH-018 using chemically synthesised reference material for the support of LC-MS/MS-based drug testing by Simon Beuck; Ines Möller; Andreas Thomas; Annika Klose; Nils Schlörer; Wilhelm Schänzer; Mario Thevis (493-505).
As recently reported, the synthetic cannabinoid JWH-018 is the subject of extensive phase I and II metabolic reactions in vivo. Since these studies were based on LC-MS/MS and/or GC-MS identification and characterisation of analytes, the explicit structural assignment of the metabolites was only of preliminary nature, if possible at all. Here, we report the chemical synthesis of five potential in vivo metabolites of JWH-018 derivatives featuring an alkylcarboxy (M1), a terminal alkylhydroxy (M2), a 5-indolehydroxy (M3), an N-dealkylated 5-indolehydroxy (M4) and a 2′-naphthylhydroxy (5) analogue, respectively, and their characterisation by nuclear magnetic resonance spectroscopy. The collision-induced dissociation (CID) patterns of the protonated compounds were studied by high-resolution/high-accuracy tandem mass spectrometry (MS n ) applying an LTQ Orbitrap with direct infusion and electrospray ionisation of target analytes. An unusual dissociation behaviour including a reversible ion–molecule reaction between a naphthalene cation (m/z 127) and water in the gas phase of the MS was shown to be responsible for nominal neutral losses of 10 u in the course of the CID pathway. LC-MS/MS-supported comparison of synthesised reference standards with an authentic urine sample using an API 4000 QTrap mass spectrometer identified the synthetic JWH-018 analogues M1–M4 as true in vivo metabolites, presuming a chromatographic separation of potentially present regioisomeric analogues. Existing doping control methods were expanded and validated according to international guidelines in order to allow for the detection of the carboxy and the alkylhydroxy metabolites, respectively, as urinary markers for the illegal intake of the synthetic cannabinoid JWH-018. Both metabolites were quantified in authentic doping control urine samples that had been suspicious of JWH-018 abuse after routine screening procedures, and a stable isotope-labelled 13C8-15N-carboxy metabolite was synthesised for future analytical applications. Figure Potential metabolites of the cannabimimetic agent JWH-018 were synthesised, characterised to probe for their in vivo generation and urinary excretion, and an LC-MS/MS-based analytical assay was validated to enable the detection of JWH-018 administration in drug testing programmes
Keywords: Spice; Synthetic cannabinoids; JWH-018; Metabolite structure; CID; Ion–molecule reaction
Determination of growth hormone releasing peptides (GHRP) and their major metabolites in human urine for doping controls by means of liquid chromatography mass spectrometry by Andreas Thomas; Sebastian Höppner; Hans Geyer; Wilhelm Schänzer; Michael Petrou; Dorota Kwiatkowska; Andrzej Pokrywka; Mario Thevis (507-516).
A family of small peptides has reached the focus of doping controls representing a comparably new strategy for cheating sportsmen. These growth hormone releasing peptides (GHRP) are orally active and induce an increased production of endogenous growth hormone (GH). While the established test for exogenous GH fails, the misuse of these prohibited substances remains unrecognized. The present study provides data for the efficient extraction of a variety of known drug candidates (GHRP-1, GHRP-2, GHRP-4, GHRP-5, GHRP-6, alexamorelin, ipamorelin, and hexarelin) from human urine with subsequent mass spectrometric detection after liquid chromatographic separation. The used method potentially enables the retrospective evaluation of the acquired data for unknown metabolites by means of a non-targeted approach with high-resolution/high-accuracy full-scan mass spectrometry with additional higher collision energy dissociation experiments. This is of great importance due to the currently unknown metabolism of most of the targets and, thus, the method is focused on the intact peptidic drugs. Only the already characterised major metabolite of GHRP-2 (d-Ala-d-2-naphthylAla-l-Ala, as well as its stable isotope-labelled analogue) was synthesised and implemented in the detection assay. Method validation for qualitative purpose was performed with respect to specificity, precision (<20%), intermediate precision (<20%), recovery (47–95%), limit of detection (0.2–1 ng/mL), linearity, ion suppression and stability. Two stable isotope-labelled internal standards were used (deuterium-labelled GHRP-4 and GHRP-2 metabolite). The proof-of-principle was obtained by the analysis of excretion study urine samples obtained from a single oral administration of 10 mg of GHRP-2. Here, the known metabolite was detectable over 20 h after administration while the intact drug was not observed.
Keywords: Illicit drugs; Sports; UPLC; Cation exchange; Non-targeted analysis
Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing by E. Solymos; S. Guddat; H. Geyer; U. Flenker; A. Thomas; J. Segura; R. Ventura; P. Platen; M. Schulte-Mattler; M. Thevis; W. Schänzer (517-528).
Methods of blood doping such as autologous and homologous blood transfusion are one of the main challenging doping practices in competitive sport. Whereas homologous blood transfusion is detectable via minor blood antigens, the detection of autologous blood transfusion is still not feasible. A promising approach to indicate homologous or autologous blood transfusion is the quantification of increased urinary levels of di(2-ethylhexyl) phthalate (DEHP) metabolites found after blood transfusion. The commonly used plasticizer for flexible PVC products, such as blood bags, is DEHP which is known to diffuse into the stored blood. Therefore, a straight forward, rapid and reliable assay is presented for the quantification of the main metabolites mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethylhexyl) phthalate that can easily be implemented into existing multi-target methods used for sports drug testing. Quantification of the DEHP metabolites was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography/tandem mass spectrometry. The method was fully validated for quantitative purposes considering the parameters specificity, linearity (1–250 ng/mL), inter- (2.4%–4.3%) and intra-day precision (0.7%–6.1%), accuracy (85%–105%), limit of detection (0.2–0.3 ng/mL), limit of quantification (1 ng/mL), stability and ion suppression effects. Urinary DEHP metabolites were measured in a control group without special exposure to DEHP (n = 100), in hospitalized patients receiving blood transfusion (n = 10), and in athletes (n = 468) being subject of routine doping controls. The investigation demonstrates that significantly increased levels of secondary DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion.
Keywords: DEHP metabolites; Sport drug testing; Urine; Liquid chromatography mass spectrometry; Blood transfusion
Urinary excretion profiles of toremifene metabolites by liquid chromatography-mass spectrometry. Towards targeted analysis to relevant metabolites in doping control by Monica Mazzarino; Xavier de la Torre; Francesco Botrè (529-541).
In the present study, toremifene urinary excretion studies were evaluated in order to examine main metabolic reactions and to select target metabolites in doping control analysis. Urine samples from three female subjects were collected every 3 h for at least 15 days after the oral administration of a single dose of Fareston® (60 mg). The elemental compositions of the compounds detected were determined by liquid chromatography-mass spectrometry using a time-of-flight system with accurate mass measurement. More detailed structure elucidation was obtained by monitoring the presence or absence of structure-specific ions, using product ion scan and neutral loss acquisition modes, whereas the metabolites urinary profiles were evaluated in selected reaction monitoring acquisition mode. The results showed that the main routes of phase-I modifications involved carboxylation of the chlorinated side chain, N-demethylation and hydroxylation in different positions. Fifteen metabolites were found in all subjects studied, most of them were detected for more than 10 days in the free, glucuronide and sulphate fractions, with a maximum of excretion generally after 9–22 and 34–47 h from drug administration. These metabolites can be divided in two groups: metabolites with the characteristic chlorine isotope pattern and metabolites without the characteristic chlorine isotope pattern. The most abundant and long-term compounds were the carboxylated metabolites followed by the hydroxylated metabolites. Their product ions originating after collision-induced dissociation were observed to occur prevalently in the dimethylaminoethoxy and in the chlorinated side chains. These structure-specific ions were used to design screening and confirmation procedures to positively identify toremifene administration in doping control analysis. Figure Suggested main metabolic routes of toremifene, as postulated by excretion studies followed by both LC-MS/MS assays with different acquisition modes and LC-QTOF
Keywords: Antidoping analysis; LC-MS; SERMs; Toremifene
Screening for benfluorex and its major urinary metabolites in routine doping controls by Mario Thevis; Gerd Sigmund; Vassilios Gougoulidis; Simon Beuck; Nils Schlörer; Andreas Thomas; Dorota Kwiatkowska; Andrzej Pokrywka; Wilhelm Schänzer (543-551).
Benfluorex [1-(m-trifluoromethylphenyl)-2-(β-benzoyloxyethyl)aminopropane] has been widely used for the treatment of atherogenic metabolic disorders and impaired carbohydrate metabolism (particularly in obese type-II diabetic patients) as well as an anorectic drug. Due to its potentially performance-enhancing properties, benfluorex has been added to the list of prohibited compounds and methods of doping by the World Anti-Doping Agency (WADA) in 2010, necessitating the implementation of the drug as well as its major metabolites into routine doping control procedures. In the present study, human urinary metabolites of benfluorex were characterized by gas chromatography–electron ionization–mass spectrometry (GC-EI-MS) as well as liquid chromatography–electrospray ionization–high resolution/high accuracy tandem mass spectrometry (LC-ESI-MS/MS). Commonly employed sports drug testing approaches consisting of liquid–liquid extraction followed by GC-MS or urine dilution and immediate LC-MS/MS analysis were expanded and validated with regard to specificity, recovery (48–54%, GC-MS only), intra- and interday precision (<25%), limits of detection (5–8 ng/mL for LC-MS/MS and 80 ng/mL for GC-MS), and ion suppression (for LC-ESI-MS/MS only) to allow the detection of benfluorex metabolites 1-(m-trifluoromethylphenyl)-2-(2-hydroxyethyl)aminopropane (M1), 1-(m-trifluoromethylphenyl)-2-(2-carboxymethyl)aminopropane (M2), and 1-(m-trifluoromethylphenyl)-2-aminopropane (M3) as well as the glucuronic acid conjugate of M1.
Keywords: Sport; Doping; Mass spectrometry; Benfluorex; Orbitrap; Stimulants
Stabilization of human urine doping control samples: a current opinion by Maria Tsivou; Dimitrios G. Georgakopoulos; Helen A. Dimopoulou; Michael Α. Koupparis; Julia Atta-Politou; Costas G. Georgakopoulos (553-561).
Transportation of doping control urine samples from the collection sites to the World Anti-doping Agency (WADA) Accredited Laboratories is conducted under ambient temperatures. When sample delivery is not immediate, microbial contamination of urine, especially in summer, is a common phenomenon that may affect sample integrity and may result in misinterpretation of analytical data. Furthermore, the possibility of intentional contamination of sports samples during collection with proteolytic enzymes, masking the abuse of prohibited proteins such as erythropoietin (EPO) and peptide hormones, is a practice that has already been reported. Consequently, stabilization of urine samples with a suitable method in a way that protects samples’ integrity is important. Currently, no stabilization method is applied in the sample collection equipment system in order to prevent degradation of urine compounds. The present work is an overview of a study, funded by WADA, on degradation and stabilization aspects of sports urine samples against the above threats of degradation. Extensive method development resulted in the creation of a mixture of chemical agents for the stabilization of urine. Evaluation of results demonstrated that the stabilization mixture could stabilize endogenous steroids, recombinant EPO, and human chorionic gonadotropin in almost the entire range of the experimental conditions tested.
Keywords: Urine stabilization; Doping-control analysis; Microorganisms; Endogenous steroids; Recombinant erythropoietin; Human chorionic gonadotropin
Specific screening method for dextran and hydroxyethyl starch in human urine by size exclusion chromatography–in-source collision-induced dissociation–time-of-flight mass spectrometry by Marjo Kolmonen; Antti Leinonen; Tiia Kuuranne; Anna Pelander; Koen Deventer; Ilkka Ojanperä (563-571).
The use of plasma volume expanders (PVE), such as dextran (DEX) and hydroxyethyl starch (HES), is prohibited in sports. DEX is a naturally occurring glucose polymer, whereas HES is synthetically produced from amylopectin starch by substitution with hydroxyethyl groups. In doping control, the commonly applied enzymatic and colorimetric screening methods are lacking adequate specificity for DEX and HES. Also, gas chromatographic–mass spectrometic (GC-MS) screening methods have specificity issues with DEX. In addition, due to the nature of the target compounds, time-consuming derivatisation steps are required in GC-MS. Based on the high molecular weight of carbohydrate polymers excreted in urine after administration of DEX and HES, a screening method was developed involving size exclusion chromatography (SEC) combined with time-of-flight mass spectrometry (TOFMS). By using solely a SEC guard column as an analytical column allowed sufficient chromatographic resolution in a minimal amount of time and with reasonable repeatability (average RSD of 10%). Detector response was linear throughout the measurement range with R 2 > 0.99 for both analytes. Limits of detection were 100 and 250 μg mL−1 for DEX and HES, respectively. Ion suppression was found to be 52% at maximum. In-source collision-induced dissociation (ISCID) was used to produce characteristic fragments at a mass accuracy better than 2 mDa. The specificity of the SEC–ISCID–TOFMS method was demonstrated with 120 PVE negative doping control samples analyzed in parallel with a routine GC-MS screening method. In addition, seven urine samples from diabetic athletes, causing interpretation problems in routine GC-MS, showed here a definitely negative profile. Figure Screening of dextran and hydroxyethyl starch in human urine by size exclusion chromatography - in-source collision induced dissociation - time-of-flight mass spectrometry
Keywords: Dextran; Hydroxyethyl starch; Screening; Size exclusion chromatography; Time-of-flight mass spectrometry
Diane Beauchemin and Dwight E. Matthews (Eds.): The encyclopedia of mass spectrometry, Volume 5: Elemental and isotope ratio mass spectrometry by Jochen Vogl (573-574).
Hugh L.J. Makin, David B. Gower (Eds.): Steroid analysis, 2nd ed. by Mario Thevis (575-576).
Letter to the Editor regarding “Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing” by Giuseppe Banfi; Massimo Franchini; Giuseppe Lippi (577-578).
Response to Letter to the Editor regarding “Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing” by E. Solymos; S. Guddat; H. Geyer; U. Flenker; A. Thomas; J. Segura; R. Ventura; P. Platen; M. Schulte-Mattler; M. Thevis; W. Schänzer (579-580).
Micro-algal biosensors by Roberta Brayner; Alain Couté; Jacques Livage; Catherine Perrette; Clémence Sicard (581-597).
Fighting against water pollution requires the ability to detect pollutants for example herbicides or heavy metals. Micro-algae that live in marine and fresh water offer a versatile solution for the construction of novel biosensors. These photosynthetic microorganisms are very sensitive to changes in their environment, enabling the detection of traces of pollutants. Three groups of micro-algae are described in this paper: chlorophyta, cyanobacteria, and diatoms.
Keywords: Biological samples; Biosensors; Fluorescence/luminescence; Metals/heavy metals; Pesticides/endocrine disruptors; Quality assurance/control
Cannabinoids and metabolites in expectorated oral fluid after 8 days of controlled around-the-clock oral THC administration by Garry Milman; Allan J. Barnes; David M. Schwope; Eugene W. Schwilke; Robert S. Goodwin; Deana L. Kelly; David A. Gorelick; Marilyn A. Huestis (599-607).
Oral fluid (OF) is an increasingly accepted matrix for drug testing programs, but questions remain about its usefulness for monitoring cannabinoids. Expectorated OF specimens (n = 360) were obtained from 10 adult daily cannabis smokers before, during, and after 37 20-mg oral Δ9-tetrahydrocannabinol (THC) doses over 9 days to characterize cannabinoid disposition in this matrix. Specimens were extracted and analyzed by gas chromatography–mass spectrometry with electron-impact ionization for THC, 11-hydroxy-THC, cannabidiol, and cannabinol, and negative chemical ionization for 11-nor-9-carboxy-THC (THCCOOH). Linear ranges for THC, 11-hydroxy-THC, and cannabidiol were 0.25–50 ng/mL; cannabinol 1–50 ng/mL; and THCCOOH 5–500 pg/mL. THCCOOH was the most prevalent analyte in 344 specimens (96.9%), with concentrations up to 1,390.3 pg/mL. 11-hydroxy-THC, cannabidiol, and cannabinol were detected in 1, 1, and 3 specimens, respectively. THC was detected in only 13.8% of specimens. The highest THC concentrations were obtained at admission (median 1.4 ng/mL, range 0.3–113.6) from previously self-administered smoked cannabis. A total of 2.5 and 3.7% of specimens were THC-positive at the recommended Substance Abuse and Mental Health Services Administration (2 ng/mL) and Driving Under the Influence of Drugs, Alcohol and Medicines (DRUID) (1 ng/mL) confirmation cutoffs, respectively. THC is currently the only analyte for monitoring cannabis exposure in OF; however, these data indicate chronic therapeutic oral THC administration and illicit oral THC use are unlikely to be identified with current guidelines. Measurement of THCCOOH may improve the detection and interpretation of OF cannabinoid tests and minimize the possibility of OF contamination from passive inhalation of cannabis smoke. Figure Median Δ9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-THC (THCCOOH) concentrations in expectorated oral fluid collected from 10 daily cannabis smokers over 9 days before, during, and after 37 20-mg oral THC doses. Arrows indicate the number of 20-mg THC doses ingested between collections
Keywords: Oral fluid; Expectoration; Cannabinoids; Δ9-tetrahydrocannabinol; Dronabinol
UHPLC-ESI-MS/MS method for direct analysis of drugs of abuse in oral fluid for DUID assessment by Sabina Strano-Rossi; Luca Anzillotti; Erika Castrignanò; Marialinda Felli; Giovanni Serpelloni; Roberto Mollica; Marcello Chiarotti (609-624).
An ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry method for the direct analysis in oral fluid (OF) of several abused drugs and metabolites in a single chromatographic run was set up and validated. Amphetamine, methamphetamine, morphine, O-6-monoacetylmorphine, cocaine, codeine, methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine, methylenedioxyamphetamine, methadone, benzoylecgonine (BEG), Δ9-tetrahydrocannabinol (THC), ketamine, and cocaethylene were determined in a single chromatographic run with no sample pretreatment, after addition of the respective deuterated internal standards. The method was designed to perform a confirmation analysis on the residual OF samples after the preliminary on-site screening test, and it was applied on preservative buffers from different devices (Mavand Rapidstat, Concateno DDS, and Greiner Bio-One) or on neat OF samples. The method was suitable to be applied to the small amounts of sample available for the confirmatory analysis after the preliminary on-site screening or on undiluted OF samples. Limits of detection varied from 5 (morphine) to 0.2 ng/mL (methamphetamine, MDMA, BEG, and cocaethylene). The method was linear for all the substances involved, giving quadratic correlation coefficients of >0.99 in all the different preservative buffers checked. In addition, repeatability and accuracy were satisfactory for the majority of the substances, except for a few cases. The developed method was subsequently applied to 466 residual samples from on-site screening performed by police officers. Of these samples, 74 showed the presence of cocaine and metabolites; THC was detected in 49 samples. Two samples showed codeine and morphine while MDMA was detected in 11 samples and ketamine in four samples.
Keywords: Forensic toxicology; Oral fluid; UHPLC-MS/MS; DUID
Quantification of carcinogenic 4- to 6-ring polycyclic aromatic hydrocarbons in human urine by solid-phase microextraction gas chromatography–isotope dilution mass spectrometry by Laura Campo; Silvia Fustinoni; PierAlberto Bertazzi (625-634).
Polycyclic aromatic hydrocarbons (PAHs) are pollutants found in living and working environments. The aim of this study was to develop a solid-phase microextraction (SPME) gas chromatography (GC)–isotope dilution mass spectrometry method for the quantification of 10 four- to six-ring PAHs in urine samples. Seven of the selected PAHs have been classified as carcinogenic. Under the final conditions, analytes were sampled with a 100-μm polydimethylsiloxane SPME fibre for 60 min at 80 °C and desorbed in the injection port of the GC at 270 °C. Fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, indeno[1,2,3-cd]pyrene and benzo[ghi]perylene were separated using a highly arylene-modified phase capillary column and quantified by MS using eight deuterated PAHs as surrogate internal standards. Limits of quantification (LOQ) were in the 0.5- to 2.2-ng/L range. Validation showed linear dynamic ranges up to 340 ng/L, inter- and intra-run precisions <20%, and accuracies within 20% of spiked concentrations. Matrix effect evaluation and the use of control charts to monitor process performances showed that the isotope dilution approach allowed for the control of bias sources. Urinary PAHs were above or equal to LOQ, depending on different compounds, in 58–100% (min–max), 40–100% and 5–39% of samples from coke oven workers (n = 12), asphalt workers (n = 10) and individuals not occupationally exposed to PAHs (n = 18), respectively. Chrysene was the most abundant PAH determined with median levels of 62.6, 6.9 and <0.6 ng/L, respectively. These results show that the method is suitable for quantifying carcinogenic PAHs in specimens from individuals with different levels of PAH exposure. Figure Single ion chromatograms for a urine sample from a coke-oven worker
Keywords: Polycyclic aromatic hydrocarbons; Urine; Solid-phase microextraction; Biological monitoring; Carcinogenic compounds
GC/MS-based metabolomic approach to validate the role of urinary sarcosine and target biomarkers for human prostate cancer by microwave-assisted derivatization by Hao Wu; Taotao Liu; Chunguang Ma; Ruyi Xue; Chunhui Deng; Huazong Zeng; Xizhong Shen (635-646).
A recent study showed that sarcosine may be potentially useful for the diagnosis and prognosis of prostate cancer (PCa). The aim of this study was to validate diagnostic value of sarcosine for PCa, to evaluate urine metabolomic profiles in patients with PCa in comparison of non-cancerous control, and to further explore the other potential metabolic biomarkers for PCa. Isotope dilution gas chromatography/mass spectrometry (ID GC/MS) metabolomic approach was applied to evaluate sarcosine using [methyl-D3]-sarcosine as an internal standard. Microwave-assisted derivatization (MAD) together with GC/MS was utilized to obtain the urinary metabolomic information in 20 PCa patients compared with eight patients with benign prostate hypertrophy and 20 healthy men. Acquired metabolomic data were analyzed using a two-sample t test. Diagnostic models for PCa were constructed using principal component analysis and were assessed with receiver–operating characteristic curves. Results showed that the urinary sarcosine level has no statistical difference between the PCa group and the control group. In addition, nine metabolomic markers between the PCa group and the healthy male group were selected, which constructed a diagnostic model with a high area under the curve value of 0.9425. We conclude that although urinary sarcosine value has limited potential in the diagnostic algorithm of PCa, urinary metabolomic panel based on GC/MS assay following MAD may potentially become a diagnostic tool for PCa.
Keywords: Metabolomic profile; Prostate cancer; Biomarker; Sarcosine; Isotope dilution gas chromatography/mass spectrometry; Microwave-assisted derivatization
Hexaplex PCR assay and liquid bead array for detection of stacked genetically modified cotton event 281-24-236×3006-210-23 by Sun Hee Choi (647-655).
A hexaplex system based on multiplex polymerase chain reaction (PCR) coupled with liquid bead array was developed to assist detection of stacked genetically modified (GM) cotton event 281-24-236 × 3006-210-23 (Widestrike) expressing two kinds of endotoxin from Bacillus thuringiensis (Bt). The efficiency of this multiplex detection system was assessed. Specific primer sets for simultaneous detection of six targets in the stacked GM cotton event were constructed and used for the PCR assay. Each of the six targets was amplified, and the amplicons could be separated as discrete bands by agarose gel electrophoresis. A liquid bead array assay for the stacked GM cotton was performed using the hexaplex PCR products followed by hybridization between the biotinylated targets and anti-tagged microsphere beads. The hybridization products produced fluorescent signals that were detected by the Luminex system. Signal strengths were analyzed by their median fluorescent intensity values. Comparison of the assays showed that results from the liquid bead array using specific probes agreed with those from the PCR, and detection of the different target elements was found to be very specific with no cross-reaction. Therefore, the combination of hexaplex PCR and liquid bead array for detection of stacked GM events can be a useful and efficient system for screening and analyzing multiple transgenes for simultaneous qualitative analysis.
Keywords: Stacked GM cotton; Hexaplex PCR; Liquid bead array; Cotton event 281-24-236 × 3006-210-23; Widestrike
Absolute protein quantification by LC-ICP-MS using MeCAT peptide labeling by Diego Esteban-Fernández; Christian Scheler; Michael W. Linscheid (657-666).
Nowadays, the most common strategies used in quantitative proteomics are based on isotope-coded labeling followed by specific molecule mass spectrometry. The implementation of inductively coupled plasma mass spectrometry (ICP-MS) for quantitative purposes can solve important drawbacks such as lack of sensitivity, structure-dependent responses, or difficulties in absolute quantification. Recently, lanthanide-containing labels as metal-coded affinity tag (MeCAT) reagents have been introduced, increasing the interest and scope of elemental mass spectrometry techniques for quantitative proteomics. In this work one of the first methodologies for absolute quantification of peptides and proteins using MeCAT labeling is presented. Liquid chromatography (LC) interfaced to ICP-MS has been used to separate and quantify labeled peptides while LC coupled to electrospray ionization mass spectrometry served for identification tasks. Synthetic-labeled peptides were used as standards to calibrate the response of the detector with compounds as close as possible to the target species. External calibration was employed as a quantification technique. The first step to apply this approach was MeCAT-Eu labeling and quantification by isotope dilution ICP-MS of the selected peptides. The standards were mixed in different concentrations and subjected to reverse-phase chromatography before ICP-MS detection to consider the column effect over the peptides. Thus, the prepared multi-peptide mix allowed a calibration curve to be obtained in a single chromatographic run, correcting possible non-quantitative elutions of the peptides from the column. The quantification strategy was successfully applied to other labeled peptides and to standard proteins such as digested lysozyme and bovine serum albumin. Figure MeCAT_Eu labeling after tryptic digestion for absolute protein quantification by LC-ICP-MS
Keywords: Quantitative proteomics; ICP-MS; MeCAT; Lanthanide labeling; Absolute quantification
Quantitative analysis of specific target DNA oligomers using a DNA-immobilized packed-column system by Seung Pil Pack; Tae-Hwe Heo; Kamakshaiah Charyulu Devarayapalli; Keisuke Makino (667-676).
Although a DNA-immobilized packed-column (DNA-packed column), which relies on sequence-dependent interactions of target DNA or mRNA (in the mobile phase) with DNA probes (on the silica particle) in a continuous flow process, could be considered as an alternative platform for quantitative analysis of specific DNA to DNA chip methodology, the performance in practice has not been satisfactory. In this study, we set up a more efficient quantitative analysis system based on a DNA-packed column by employing a temperature-gradient strategy and DMSO-containing mobile phase. Using a temperature-gradient strategy based on T m values of probe/target DNA hybridizations and DMSO (5%)-containing mobile phase, we succeeded in the quantitative analysis of a specific complementary target distinguishable from non-complementary DNA oligomers or other similar DNA samples. In addition, two different target DNA oligomers even with similar T m values were separated and detected quantitatively by using a packed column carrying two different DNA probes.
Keywords: Bioanalytical methods; Chromatography; DNA-immobilized packed column; DNA oligomer separation
Investigation on the phenolic constituents in Hamamelis virginiana leaves by HPLC-DAD and LC-MS/MS by Sarina M. Duckstein; Florian C. Stintzing (677-688).
Aqueous and acetone/water extracts from Hamamelis virginiana leaves were investigated to obtain a thorough insight into their phenolic composition. To secure compound integrity, a gentle extraction method including the exclusion of light was used. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses yielded a fingerprint including 27 phenolic constituents. Quantification of the key compounds on an equivalent basis by high-performance liquid chromatography diode-array detection (HPLC-DAD) showed that gallotannins consisting of six to 11 galloyl units constitute the main fraction, whereas procyanidins and catechin represented only a minor part. Closer inspection revealed that both extracts possess virtually the same galloyl hexose distribution, and the octagalloyl hexose represents the major tannin constituent. Additionally, eight flavonol glycosides and their corresponding aglycones quercetin and kaempferol, as well as three chlorogenic acid isomers and other hydroxycinnamic acids, were identified. Moreover, stability studies on the aqueous extract (5 °C, dark; room temperature, dark; room temperature, light) revealed that the phenolic profile underwent changes when exposed to light. Especially the gallotannins proved to be considerably unstable which may result in phytochemically altered Hamamelis leaf extracts upon transport and storage. Figure Phenolic constituents in Hamamelis.virginiana leaves
Keywords: Hamamelis virginiana ; Gallotannins; Chlorogenic acids; Flavonol glycosides; HPLC-DAD-MS/MS; Stability
Effect of d-allose on prostate cancer cell lines: phospholipid profiling by nanoflow liquid chromatography–tandem mass spectrometry by Rae Ung Jeong; Sangsoo Lim; Myoung Ok Kim; Myeong Hee Moon (689-698).
d-Allose, a rare, naturally occurring monosaccharide, is known to exert anti-proliferative effects on cancer cells. The effects of d-allose on the cellular membranes of hormone-refractory prostate cancer cell line (DU145), hormone-sensitive prostate cancer cell line (LNCaP), and normal prostate epithelial cells (PrEC) were studied at the molecular level by phospholipid (PL) profiling using a shotgun lipidomic method. The molecular structures of 85 PL species including 23 phosphatidylcholines, 12 phosphatidylethanolamines (PEs), 11 phosphatidylserines (PSs), 16 phosphatidylinositols, 9 phosphatidic acids (PAs), and 14 phosphatidylglycerols (PGs) were identified by data-dependent collision-induced dissociation of nanoflow liquid chromatography–tandem mass spectrometry, and the PL amounts were quantified. The addition of d-allose to prostate cancer cell lines during their growth phases had negligible or decreased effects on the relative regulation of PL species, but several new PS molecules (two for DU145 and three for LNCaP) emerged. In contrast, experiments on the PrEC cell line revealed that some high abundant species (14:0/14:0-PE, 16:2/16:0-PG, and 20:6/18:1-PA) showed significant increases in concentration. These findings support a mechanism for the anti-proliferative effect of d-allose on prostate cancer cell lines that involves the induction of programmed cell death since PS molecules are known to induce apoptosis. Principal component analysis was carried out to examine differences in PL distributions among the three cell lines promoted by d-allose.
Keywords: Phospholipids; nLC-ESI-MS-MS; Prostate cancer cell lines; Effect of d-Allose; HRPC cells
A new aminopeptidase inhibitor from Streptomyces strain HCCB10043 found by UPLC–MS by Min Rao; Qiushuang Li; Lei Feng; Xing Xia; Lijun Ruan; Xiafang Sheng; Mei Ge (699-706).
The diversity of microbial metabolites has been of interest and concern for a long time, yet a suitable method for discovering these is still unavailable. In the work discussed in this report, ultra-performance liquid chromatography coupled with tandem quadrupole and time of flight high-resolution mass spectrometry (UPLC–Q–TOF-HRMS), with MS data analysis, was set up to study the metabolites of Streptomyces strain HCCB10043. It was found that besides antibacterial substances (A21978C complex) and two anti-aminopeptidase compounds (valistatin and bestatin), this strain can produce a new aminopeptidase inhibitor, identified as 3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine. This new compound had greater activity than valistatin or bestatin in aminopeptidase N (APN) inhibition assay. The results proved that combination of UPLC–Q–TOF-MS analysis and classic purification and identification steps as complementary strategies can provide a method with high reliability for research on microbial secondary metabolites. Furthermore, it has shown that the study of secondary metabolic profiling might be the key to discovering new drugs. Figure Pathway to the new aminopeptidase inhibitor
Keywords: Diversity; UPLC–Q–TOF-HRMS; Streptomyces sp. HCCB10043; Aminopeptidase inhibitor
Characterization and application of a diamine oxidase from Lathyrus sativus as component of an electrochemical biosensor for the determination of biogenic amines in wine and beer by Massimo Di Fusco; Rodolfo Federico; Alberto Boffi; Alberto Macone; Gabriele Favero; Franco Mazzei (707-716).
In this work, we have characterized a diamine oxidase (DAO) from Lathyrus sativus and evaluated its use, for the first time, as biocatalytic component of an electrochemical biosensor for the determination of biogenic amines index in wine and beer samples. Firstly, DAO was electrokinetically characterized free in solution by means of a platinum electrode and then immobilized by using polyazetidine prepolimer on the surface of screen-printed electrodes constituted of two gold working electrodes. The amperometric measurements were carried out by using a flow system at a fixed potential of +600 mV vs the internal silver pseudo reference in phosphate buffer solution (0.1 mol l-1, pH = 7.4). The analysis of wine and beer samples were performed in flow injection system using the dual channel transducer providing simultaneous detection of sample and blank signal, and the resulting signal (after subtraction of the blank signal) was referred to that of putrescine. The results were compared with those obtained using a modified reference method based on gas chromatography-mass spectrometry analysis on the same samples. The results obtained in the analysis of Italian wines shows the better suitability of DAO-based biosensor in the determination of the biogenic amines (BAs) index expressed as putrescine equivalent in both red and white wines, being less efficient in beer samples where it underestimates by about 50% the BAs content.
Keywords: Diamine oxidase; Biogenic amines index; Biosensors; Wine
Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum by Sabine Nutz; Katharina Döll; Petr Karlovsky (717-726).
Real-time PCR (qPCR) is the principal technique for the quantification of pathogen biomass in host tissue, yet no generic methods exist for the determination of the limit of quantification (LOQ) and the limit of detection (LOD) in qPCR. We suggest using the Youden index in the context of the receiver operating characteristic (ROC) curve analysis for this purpose. The LOQ was defined as the amount of target DNA that maximizes the sum of sensitivity and specificity. The LOD was defined as the lowest amount of target DNA that was amplified with a false-negative rate below a given threshold. We applied this concept to qPCR assays for Fusarium verticillioides and Fusarium proliferatum DNA in maize kernels. Spiked matrix and field samples characterized by melting curve analysis of PCR products were used as the source of true positives and true negatives. On the basis of the analysis of sensitivity and specificity of the assays, we estimated the LOQ values as 0.11 pg of DNA for spiked matrix and 0.62 pg of DNA for field samples for F. verticillioides. The LOQ values for F. proliferatum were 0.03 pg for spiked matrix and 0.24 pg for field samples. The mean LOQ values correspond to approximately eight genomes for F. verticillioides and three genomes for F. proliferatum. We demonstrated that the ROC analysis concept, developed for qualitative diagnostics, can be used for the determination of performance parameters of quantitative PCR.
Keywords: Real-time PCR; Fusarium verticillioides ; Fusarium proliferatum ; Receiver operating characteristic; Limit of detection; Limit of quantification
Simultaneous determination of albendazole and its metabolites in fish muscle tissue by stable isotope dilution ultra-performance liquid chromatography tandem mass spectrometry by Xiaojun Zhang; Hanxiang Xu; Hong Zhang; Yuanming Guo; Zhiyuan Dai; Xuechang Chen (727-734).
A rapid, specific, and sensitive method utilizing ultra-performance liquid chromatography tandem mass spectrometry was developed and validated to determine albendazole, albendazole sulfoxide, albendazole sulfone, and albendazole 2-aminosulfone in fish muscle tissue. The fish samples were extracted with ethyl acetate, then the organic phase was evaporated to dryness, and the residue was reconstituted in methanol–water solution and cleaned up by n-hexane. Reversed-phase separation of target compounds was achieved using a BEH C18 column and a gradient consisting of 0.2% (v/v) formic acid and methanol. Tandem mass spectrometry analyses were performed on a triple–quadrupole tandem mass spectrometer. In the whole procedure, the isotope-labeled internal standards were used to correct the matrix effect and variations associated with the analysis. The method was validated with respect to linearity, specificity, accuracy, and precision. The method exhibited a linear response from 0.1 to 20 ng mL-1 (r 2 > 0.9985). The limit of quantitation for albendazole (ABZ), albendazole sulfoxide (ABZSO), albendazole sulfone (ABZSO2), and albendazole 2-aminosulfone (ABZ-2-NH2SO2) was 0.1, 0.1, 0.1, and 0.2 ng g-1, respectively. The mean recoveries of ABZ, ABZSO, ABZSO2, and ABZ-2-NH2SO2 spiked at a level of 0.2–5.0 ng g-1 were 95.3–113.7%, and the relative standard deviations of intra- and inter-day measurements were less than 6.38%. The method was later successfully applied to the determination of albendazole and its three metabolites in 60 fish samples collected from local markets. Figure Chromatogram of albendazole and its metabolites
Keywords: UPLC-MS/MS; Albendazole; Albendazole sulfoxide; Albendazole sulfone; Albendazole 2-aminosulfone; Fish muscle
Analysis of natural red dyes (cochineal) in textiles of historical importance using HPLC and multivariate data analysis by Ana Serrano; Micaela M. Sousa; Jessica Hallett; João A. Lopes; M. Conceição Oliveira (735-743).
A new analytical approach based on high-performance liquid chromatography with diode array detector (HPLC-DAD) and multivariate data analysis was applied and assessed for analyzing the red dye extracted from cochineal insects, used in precious historical textiles. The most widely used method of analysis involves quantification of specific minor compounds (markers), using HPLC-DAD. However, variation in the cochineal markers concentration, use of aggressive dye extraction methods and poor resolution of HPLC chromatograms can compromise the identification of the precise insect species used in the textiles. In this study, a soft extraction method combined with a new dye recovery treatment was developed, capable of yielding HPLC chromatograms with good resolution, for the first time, for historical cochineal-dyed textiles. After principal components analysis (PCA) and mass spectrometry (MS), it was possible to identify the cochineal species used in these textiles, in contrast to the accepted method of analysis. In order to compare both methodologies, 7 cochineal species and 63 historical cochineal insect specimens were analyzed using the two approaches, and then compared with the results for 15 historical textiles in order to assess their applicability to real complex samples. The methodology developed here was shown to provide more accurate and consistent information than the traditional method. Almost all of the historical textiles were dyed with Porphyrophora sp. insects. These results emphasize the importance of adopting the proposed methodology for future research on cochineal (and related red dyes). Mild extraction methods and HPLC-DAD/MSn analysis yield distinctive profiles, which, in combination with a PCA reference database, are a powerful tool for identifying red insect dyes. Figure In pursuit of a precious red dye! A new methodology has been developed for determining the precise cochineal dye used in historical textiles. Mild extraction methods and HPLC-DAD yield distinctive profiles that, in combination with a dye reference database based on PCA, creates a powerful tool for identifying the precise red dye used. Surprisingly, almost all of the historical textiles analysed were not dyed with American cochineal.
Keywords: Cochineal; Dyes/Pigments; HPLC-DAD-UV; MS; Multivariate data analysis; Natural dyestuffs
Quantitative multi-element mapping of ancient glass using a simple and robust LA-ICP-MS rastering procedure in combination with image analysis by Vid S. Šelih; Johannes T. van Elteren (745-755).
The surface of two glass artefacts in mosaic style, probably fragments of conglomerate glass bowls dating back two millennia, was investigated by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS). By rastering with the laser beam over a selected area of the surface of the glass artefacts, elemental oxide maps were generated. Quantification of the elemental oxides in the maps was achieved using a so-called sum normalization procedure, summating the elements—54 in total—as their oxides to 100% (w/w), without using an internal standard and applying only one external standard (NIST SRM glass 610). This results in a robust mapping procedure which automatically corrects for drift and defocusing issues. Sum normalization was applied to each pixel in the map separately and required a custom source code to process all the data in the tens of thousands of pixels to generate the elemental oxide concentration maps. The digital element maps generated upon rastering of the two glass artefacts are very compelling and are an excellent entry point to gain detailed insight into their fabrication and provenance using image analysis software for retrieval of localized elemental oxide concentrations and correlations. Figure Investigated decorative glass artefact, probably a fragment of a glass bowl dating back two millenia
Keywords: Mosaic glass; Elemental composition; Mapping; Image analysis; LA-ICP-MS
Terracotta polychrome sculptures examined before and after their conservation work: contributions from non-invasive in situ analytical techniques by C. Colombo; F. Bevilacqua; L. Brambilla; C. Conti; M. Realini; J. Striova; G. Zerbi (757-765).
The potential of non-invasive in situ analytical techniques such as portable Raman, portable X-ray fluorescence, portable optical microscope and fibre optics reflectance spectroscopy has been shown studying painted layers of Renaissance terracotta polychrome sculptures belonging to the statuary of Santo Sepolcro Church in Milan. The results obtained allowed pointing out the contribution of these techniques to the compositional diagnostic, providing complete information, in some cases, better than micro-destructive techniques, on the kind of pigments used on the external painted layers. Moreover, a comparison with the results obtained before the last conservation work (2009) with micro-destructive techniques allowed ascertaining the removal of the external painted layers during the conservation operations.
Keywords: Portable XRF; Portable Raman; FORS; Polychrome terracotta statues; Monitoring
Liquid chromatography time-of-flight mass spectrometry following sorptive microextraction for the determination of fungicide residues in wine by A. R. Fontana; I. Rodríguez; M. Ramil; J. C. Altamirano; R. Cela (767-775).
This work evaluates the suitability of sorptive microextraction, using disposable silicone sorbents, and liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) for the determination of 15 fungicides in wine. Under optimized conditions, wine samples (10 mL) were diluted with the same volume of ultrapure water and poured in a glass vessel containing a magnetic stirrer and 4 g of sodium chloride. Extractions were performed at room temperature for 4 h, using an inexpensive silicone disk (12 μL volume) exposed directly to the sample. Thereafter, analytes were recovered with 0.2 mL of acetonitrile. The electrospray ionization (ESI) source was operated in the fast polarity switching mode obtaining, in the same injection, selective LC-MS records (extracted with a mass window of 10 ppm) of compounds rendering [M + H]+ and [M-H]− ions. The method provided limits of quantification (LOQs) between 0.1 and 2.2 ng mL−1, linear response ranges up to 500 ng mL−1, relative recoveries from 75% to 117% and an inter-day variability below 15% for all analytes in red and white wine samples. The feasibility of in situ sample enrichment followed by delayed desorption and analysis is also assessed.
Keywords: Sorptive microextraction; Silicone sorbents; Fungicides; Wine; Liquid chromatography; Time-of-flight mass spectrometry
Optical waveguides for the evanescent wave-induced cleavage of photolabile linker compounds by Katrin Schmitt; Jonas Rist; Christian Hoffmann (777-782).
Functional surfaces and especially the control of surface properties depending on external parameters such as light illumination have gained increasing importance in the last few years. We present the characterization of polymers from the cycloolefin (co)polymer class (COC/COP) functionalized with an aminosilane as a basis for the further immobilization of compounds. In a first step, an assay using AlexaFluor®647 fluorescent dye was used to assess surface homogeneity and reproducibility. A coefficient of variation of less than 15% for dot-to-dot and less than 25% for chip-to-chip could be achieved. The same amino-functionalized surfaces were then used to immobilize a biotinylated photolabile linker compound, binding AlexaFluor®647-labeled streptavidin. The linker was photocleaved with high efficiency at λ = 365 nm and P = 0.15 mW/cm2. Fluorescence measurements show that polymers of the COC/COP class can be used as versatile surfaces for the photoinduced release of compounds immobilized via photolabile linkers. Figure Photo-induced release of a model compound
Keywords: COC/COP polymer; Photolabile linker; Fluorescence; Evanescent field