Analytical and Bioanalytical Chemistry (v.401, #6)

Focus on analytical chemistry to illuminate the past by Maria Perla Colombini (1725-1726).
is currently Full Professor of Analytical Chemistry at the Department of Chemistry, Faculty of Natural, Mathematical and Physical Sciences of the University of Pisa. Her research focuses on the development of analytical procedures based on mass spectrometric and chromatographic techniques for characterisation of natural and synthetic organic materials (e.g. proteinaceous binders, siccative oils, vegetable resins, colorants, gums) and their degradation products in works of art and archaeological objects. Her research has resulted in over 160 publications in refereed journals and she is the editor of three books. She is head of a Master’s in “Material and diagnostic technique in cultural heritage” and is currently President of the Italian Archaeometry Association.

Discovering the composition of ancient cosmetics and remedies: analytical techniques and materials by Erika Ribechini; Francesca Modugno; Josefina Pérez-Arantegui; Maria Perla Colombini (1727-1738).
This article reviews the analytical techniques and procedures used in the study of ancient cosmetics, therapeutic chemicals, and remedies found in historical and archaeological sites. Well consolidated techniques based on molecular and atomic spectroscopy, for example FT-IR, Raman, SEM-EDX, and XRD, and analytical procedures based on high-performance chromatography and mass spectrometry, for example GC–MS and HPLC–MS are reviewed. The advantages of recently introduced techniques based on synchrotron radiation and on direct mass spectrometric techniques are also discussed. The possibility of extracting information about composition, preparation techniques, and the degradation processes of ancient cosmetics, pharmaceutics, and ritual balms is analysed by use of several case studies. Figure
Keywords: Ancient cosmetics and remedies; Molecular and atomic spectroscopy; Synchrotron radiation; High performance chromatography; Mass spectrometry

First chemical evidence of royal purple as a material used for funeral treatment discovered in a Gallo–Roman burial (Naintré, France, third century AD) by Thibaut Devièse; Erika Ribechini; Pietro Baraldi; Bernard Farago-Szekeres; Henri Duday; Martine Regert; Maria Perla Colombini (1739-1748).
Violet–purple residues collected from a Gallo–Roman burial dated back to the second half of the third century A.D. and excavated at Naintré (France) were chemically investigated by multi-analytical methodology involving the use of Raman spectroscopy, direct exposure-mass spectrometry (DE-MS) and high-performance liquid chromatography (HPLC–UV–visible). Little is known about funeral treatment and rituals during Roman times. Retrieving valuable information on these by chemical analysis of organic residues was thus a key aspect of this work. Analyses demonstrated the presence of the very precious purple colorant obtained from shellfish glands commonly known as Tyrian or royal purple and its exceptional preservation. Chemical investigation and archaeological evidence have shown that purple was widely spread after the deposition of the body for burial. These results are the earliest chemical evidence of purple colorant used during funeral rituals (not as textile dye) and enabled us to highlight new aspects of funeral practices in Roman times.
Keywords: Purple; Roman burial; Funeral treatment; Raman spectroscopy; HPLC; Direct exposure-mass spectrometry

Residues from medicine containers in the collections of the British Museum have been investigated as part of a wider programme of scientific work on Roman surgical instruments. The cylindrical bronze containers are often described as instrument cases, but some contain materia medica, ranging from extensive extant remains of ancient preparations to possible minor deposits on the interior surfaces of the containers. Samples from seven residues have been analysed by gas chromatography–mass spectrometry (GC-MS) to identify lipid, resin and carbohydrate components and by X-ray fluorescence and Raman spectroscopy to characterise inorganic materials. The results have provided evidence for ointments and powders or pills consistent with a medical purpose. The ingredients identified include beeswax, fat, conifer resin and gum-derived sugars, plus elemental carbon and lead and zinc salts. Particularly significant were the varied compositions of residues from four sections of a multi-compartment container. In one of these compartments, the beeswax seems to have been prepared as the ‘Punic wax’ described by Pliny. Experimental preparation of Punic wax following Pliny’s method was undertaken in the laboratory and the product analysed to compare with the ointment residues. This paper discusses the GC-MS results of both the experimental material and the archaeological residues and their significance for the interpretation of the past intended applications of the medicines and the use of the containers.
Keywords: Medicine; Archaeology; Beeswax; Punic wax; Residues; Saponification; GC-MS; XRF

Pyrolysis–GC/MS for the identification of macromolecular components in historical recipes by Chiara Riedo; Dominique Scalarone; Oscar Chiantore (1761-1769).
Analytical pyrolysis with thermally assisted hydrolysis and methylation was employed to investigate ancient ointments collected from Spanish vessels coming from the sixteenth century pharmacies. The ointments were reproduced on the basis of historical recipes and characterization was made in comparison with real samples. Characteristic markers indicate the presence of beeswax, of animal and plant lipids, and of natural resins. Analyses of old samples are consistent with the modern reproductions and with the analysis of raw materials. Multivariate data analysis was used to discriminate between the different types of lipidic materials, also in connection with their relative amount in the samples. Figure Score plot of principal component analysis performed on data obtained from real samples, reference ointments, and raw materials by means of thermally assisted hydrolysis and methylation
Keywords: Py–GC/MS; Ointments; Historical recipes; Thermally assisted hydrolysis and methylation; Lipids; Resins

Historical nomenclature has not always been unequivocally associated with the botanical origin of natural resins. The availability of natural resins has changed throughout the centuries and so have their trade names. Furthermore, adulterations and lack of knowledge have led to variations in the composition of the products traded under the same name. This investigation aims at a new understanding of the interrelation between the historical and modern terms for natural resins. Different Pinaceae and Pistacia resins, mastic, sandarac, copaiba balm and burgundy pitch from Vigani’s Cabinet, a 300-year-old pharmaceutical collection owned by Queens’ College, Cambridge (UK) were investigated. Related reference materials from modern collections were analysed together with natural resins derived from reliable botanical sources. The analytical method was gas chromatography/mass spectrometry (GC-MS) with and without derivatisation with trimethylsulfonium hydroxide. This technique provided detailed molecular compositions of the studied materials, which in turn led to particular data profiles of the materials. Marker molecules of Copaifera, Pinaceae, Cupressaceae and Pistacia resins were identified. By comparing the GC-MS data profiles to the reference samples, a clearer picture of the connection between nomenclature and botanical origin was obtained. With the aid of the marker molecules and data profiles, it was then possible to clarify the nomenclature of the aged resins sampled from Vigani’s Cabinet. Figure Four drawers from the Vigani Cabinet
Keywords: Diterpenoid; Triterpenoid; Resin; Gas chromatography–mass spectrometry; trimethylsulfonium hydroxide; Eighteenth century; Material collection; Botanical source; John Francis Vigani

HPLC–APCI-MS analysis of triacylglycerols (TAGs) in historical pharmaceutical ointments from the eighteenth century by Francesco Saliu; Francesca Modugno; Marco Orlandi; Maria Perla Colombini (1785-1800).
The lipid fractions of residues from historical pharmaceutical ointments were analysed by reversed-phase liquid chromatography coupled with atmospheric pressure chemical ionization and mass spectrometer detection. The residues were contained in a series of historical apothecary jars, dating from the eighteenth century and conserved at the “Aboca Museum” in Sansepolcro (Arezzo, Italy) and at the pharmacy of the “Real Cartuja de Valldemossa” in Palma de Majorca (Spain). The analytical protocol was set up using a comparative study based on the evaluation of triacylglycerol (TAG) compositions in raw natural lipid materials and in laboratory-reproduced ointments. These ointments were prepared following pharmaceutical recipes reported in historical treatises and used as reference materials. The reference materials were also subjected to stress treatments in order to evaluate the modification occurring in the TAG profiles as an effect of ageing. TAGs were successfully detected in the reproduced formulations even in mixtures of up to ten ingredients and after harsh degradative treatments, and also in real historical samples. No particular interferences were detected from other non-lipid ingredients of the formulations. The TAG compositions detected in the historical ointments indicated a predominant use of olive oil and pig adipose material as lipid ingredients. The detection of a high level of tristearine and myristyl-palmitoyl-stearyl glycerol in two of the samples suggested the presence of a fatty material of a different origin (maybe a ruminant). On the basis of the positional isomer ratio, sn-PPO/sn-POP, it was possible to hypothesize an exclusive use of pig fat in one sample. We also evaluated the application of principal component analysis of TAG profiles as an approach for the multivariate statistical comparison of the reference and historical ointments. Figure HPLC-APCI-MS analysis of triacylglycerols (TAGs) in historical pharmaceutical ointments from the eighteenth century
Keywords: Archaeometry; Ointments; Triacyilglycerols; APCI-MS

A multi-analytical approach for the characterization of powders from the Pompeii archaeological site by Carmen Canevali; Paolo Gentile; Marco Orlandi; Francesca Modugno; Jeannette Jacqueline Lucejko; Maria Perla Colombini; Laura Brambilla; Sara Goidanich; Chiara Riedo; Oscar Chiantore; Pietro Baraldi; Cecilia Baraldi; Maria Cristina Gamberini (1801-1814).
Nine black powders found in Pompeii houses in three different types of bronze vessels (cylindrical theca atramentaria, unguentaries, and aryballoi) were characterized in order to assess a correspondence between the composition and the type of vessel and, possibly, to verify if these powders were inks or not. For the compositional characterization, a multi-analytical approach was adopted, which involved the use of scanning electron microscopy–energy dispersive X-ray, Fourier-transformed infrared spectroscopy, Raman, X-ray diffraction, electron paramagnetic resonance spectroscopy, thermogravimetric analysis, gas chromatography coupled with mass spectrometry (GC/MS), and pyrolysis GC/MS. Powders contained in cylindrical theca atramentaria form a homogeneous group, and their organic and inorganic compositions suggest that they were writing inks, while powders contained in unguentaries and aryballoi could have had several different uses, including writing inks and cosmetics. Furthermore, the composition profile of the powders found in cylindrical cases shows that, at 79 ad, in Pompeii, carbon-based inks were still used for writing, and iron gall inks had not been introduced yet. Figure Photography of the 12724 black powder contained in a cylindrical theca atramentaria.
Keywords: SEM-EDX; FTIR; RAMAN; XRD; EPR; GC/MS

Non-invasive and micro-destructive investigation of the Domus Aurea wall painting decorations by Catia Clementi; Valeria Ciocan; Manuela Vagnini; Brenda Doherty; Marisa Laurenzi Tabasso; Cinzia Conti; Brunetto Giovanni Brunetti; Costanza Miliani (1815-1826).
The paper reports on the exploitation of an educated multi-technique analytical approach based on a wide non invasive step followed by a focused micro-destructive step, aimed at the minimally invasive identification of the pigments decorating the ceiling of the Gilded Vault of the Domus Aurea in Rome. The combination of elemental analysis with molecular characterization provided by X-ray fluorescence and UV–vis spectroscopies, respectively, allowed for the in situ non-invasive identification of a remarkable number of pigments, namely Egyptian blue, green earth, cinnabar, red ochre and an anthraquinonic lake. The study was completed with the Raman analysis of two bulk samples, in particular, SERS measurements allowed for the speciation of the anthraquinonic pigment. Elemental mapping by scanning electron microscopy-energy dispersive spectrometer combined with micro-fluorimetry on cross-section gave an insight into both the distribution of different blend of pigments and on the nature of the inorganic support of the red dye.
Keywords: Non-invasive analysis; SERS; Roman wall painting; Egyptian blue; Anthraquinonic lake

Characterization of fresh and aged natural ingredients used in historical ointments by molecular spectroscopic techniques: IR, Raman and fluorescence by L. Brambilla; C. Riedo; C. Baraldi; A. Nevin; M. C. Gamberini; C. D’Andrea; O. Chiantore; S. Goidanich; L. Toniolo (1827-1837).
Natural organic materials used to prepare pharmaceutical mixtures including ointments and balsams have been characterized by a combined non-destructive spectroscopic analytical approach. Three classes of materials which include vegetable oils (olive, almond and palm tree), gums (Arabic and Tragacanth) and beeswax are considered in this study according to their widespread use reported in ancient recipes. Micro-FTIR, micro-Raman and fluorescence spectroscopies have been applied to fresh and mildly thermally aged samples. Vibrational characterization of these organic compounds is reported together with tabulated frequencies, highlighting all spectral features and changes in spectra which occur following artificial aging. Synchronous fluorescence spectroscopy has been shown to be particularly useful for the assessment of changes in oils after aging; spectral difference between Tragacanth and Arabic gum could be due to variations in origin and processing of raw materials. Analysis of these materials using non-destructive spectroscopic techniques provided important analytical information which could be used to guide further study.
Keywords: Oils; Gums; Beeswax; FTIR; Raman; Fluorescence; Non-destructive analysis

A study of the composition of the remains of ancient ointments from museums was undertaken to enable understanding of the preparation techniques. Comparison of ancient recipes from different historical periods and spectroscopic characteristics of inorganic and/or organic remains recovered in museum vessels enabled preparation of ancient pharmaceutical–cosmetic formulations. Farmacopea Augustana by Occo was one the most important books studied for the 14 formulations prepared in the laboratory. Three formulations are discussed in detail and raw materials and new preparations were proposed for ozone ageing. The most important micro Raman results are discussed. The spectra of the raw materials lipids, beeswax, and resins are discussed; beeswax and pig suet (axŭngia) Raman spectra were found to be similar, but different from those of the aged oils. SERS was applied to ancient ointments and galbanum and the Raman spectra are reported and discussed for the first time. Figure Optical appearances of ancient recipes, ointments and cosmetic/pharmaceutical laboratory preparations.
Keywords: Recipe books; Pharmacopoeia; Ointments; Raman spectroscopy; SERS

A round robin exercise in archaeometry: analysis of a blind sample reproducing a seventeenth century pharmaceutical ointment by M. P. Colombini; F. Modugno; M. C. Gamberini; M. Rocchi; C. Baraldi; T. Deviese; R. J. Stacey; M. Orlandi; F. Saliu; C. Riedo; O. Chiantore; G. Sciutto; E. Catelli; L. Brambilla; L. Toniolo; C. Miliani; P. Rocchi; J. Bleton; U. Baumer; P. Dietemann; G. Pojana; S. Marras (1847-1860).
Chemical analysis of ancient residues of pharmaceutical or cosmetic preparations such as balms or ointments is made problematic by the high complexity of these mixtures, composed of organic and inorganic materials. Consequently, a multi-analytical approach and special caution in the interpretation of the results are necessary. In order to contribute to the improvement of analytical strategies for the characterization of complex residues and to reconstruct ancient medical practices, a replica of a pharmaceutical formulation of the seventeenth century was prepared in the laboratory according to a historically documented recipe. In a round robin exercise, a portion of the preparation was analysed as a blind sample by 11 laboratories using various analytical techniques. These included spectroscopic, chromatographic and mass spectrometric methods. None of the laboratories was able to completely reconstruct the complex formulation, but each of them gave partial positive results. The round robin exercise has demonstrated that the application of a multi-analytical approach can permit a complete and reliable reconstruction of the composition. Finally, on the basis of the results, an analytical protocol for the study of residues of ancient medical and pharmaceutical preparations has been outlined.
Keywords: Round robin; Blind sample; Ointment; Analytical techniques

A zebrafish scale assay to monitor dioxin-like activity in surface water samples by Sergi Pelayo; Ramón López-Roldán; Susana González; Marta Casado; Demetrio Raldúa; Jose Luis Cortina; Benjamin Piña (1861-1869).
New regulations on water quality require a close control of the possible biological activities known or unexpected pollutants may bring about. We present here a protocol based on the direct exposure of zebrafish to river water and the analysis of expression of specific genes in their scales to determine the presence of compounds with dioxin-like biological activity. The method does not require the killing of animals and allows detection of the biological activity after a single day of exposure. When tested, the method with real samples from the Llobregat River, clear temporal and spatial variations were observed, demonstrating its suitability for monitoring natural variations in water quality linked to specific discharges. High biological activities were unrelated to the currently checked water quality parameters (macropollutants, turbidity, TOC, etc.), but they did correlate with the presence of micropollutants (estrogens, detergents, etc.) related to domestic and/or industrial runoffs. The scale assay therefore provides a new tool to evaluate water quality changes that cannot be easily derived from the existing standard analytical procedures. It ranks among the very few described protocols able to detect biological effects from natural water samples, without a pre-concentration step, and after only 24 h of exposure.
Keywords: Dioxine-like pollutants; Real-time PCR; Danio rerio ; Bioassays; RNA quantification; cyp1a; GC-MS

Potential analytical applications of lysenin channels for detection of multivalent ions by Daniel Fologea; Redwan Al Faori; Eric Krueger; Yuriy I. Mazur; Matt Kern; Matt Williams; Amir Mortazavi; Ralph Henry; Greg J. Salamo (1871-1879).
Transmembrane protein transporters possessing binding sites for ions, toxins, pharmaceutical drugs, and other molecules constitute excellent candidates for developing sensitive and selective biosensing devices. Their attractiveness for analytical purposes is enhanced by the intrinsic amplification capabilities shown when the binding event leads to major changes in the transportation of ions or molecules other than the analyte itself. The large-scale implementation of such transmembrane proteins in biosensing devices is limited by the difficulties encountered in inserting functional transporters into artificial bilayer lipid membranes and by the limitations in understanding and exploiting the changes induced by the interaction with the analyte for sensing purposes. Here, we show that lysenin, a pore-forming toxin extracted from earthworm Eisenia foetida, which inserts stable and large conductance channels into artificial bilayer lipid membranes, functions as a multivalent ion-sensing device. The analytical response consists of concentration and ionic-species-dependent macroscopic conductance inhibition most probably linked to a ligand-induced gating mechanism. Multivalent ion removal by chelation or precipitation restores, in most cases, the initial conductance and demonstrates reversibility. Changes in lipid bilayer membrane compositions leading to the absence of voltage-induced gating do not affect the analytical response to multivalent ions. Microscopic current analysis performed on individual lysenin channels in the presence of Cu2+ revealed complex open–closed transitions characterized by unstable intermediate sub-conducting states. Lysenin channels provide an analytical tool with a built-in sensing mechanism for inorganic and organic multivalent ions, and the excellent stability in an artificial environment recommend lysenin as a potential candidate for single-molecule detection and analysis.
Keywords: Biosensors; Multivalent ions; Lysenin; Pore-forming toxins; Bilayer lipid membranes

Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives by Weigang Huang; Dechen Jiang; Xiaoyang Wang; Kelong Wang; Christopher E. Sims; Nancy L. Allbritton; Qisheng Zhang (1881-1888).
Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP2 derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications. Online abstract Fluorescent PIP2 is efficiently phosphorylated by PI3K to form fluorescent PIP3. Consequently, PI3K activity can be measured by separating and quantifying fluorescent PIP2 and PIP3
Keywords: PI3K assay; Fluorescent phosphatidylinositides; TLC analysis; Capillary electrophoresis

Process for detecting Helicobacter pylori using aliphatic amides by José A. Ferreira; Elsa Dias; Sílvia M. Rocha; Manuel A. Coimbra (1889-1898).
Helicobacter pylori diagnosis is fundamental in the management of gastrointestinal pathologies, whose current clinical guidelines support a non-invasive ‘test-and-treat’ strategy. As such, the present work reports the basis of a new, low-cost, specific breath test based on the detection of volatile carboxylic acids resulting from the hydrolysis of short-chain aliphatic amides by H. pylori amidases. Propionamide and butyramide, which are metabolized by amidases to propionic and butyric acids, were elected for this study. Conditions for the extraction of these acids from a vapour phase were optimized concerning the use of solid-phase microextraction (SPME) followed by gas chromatography–quadrupole mass spectrometry (GC–qMS) analysis. SPME–GC–qMS was then used to detect the acids released into a vapour phase upon incubation of a H. pylori reference strain J99 or a clinical specimen with the amides. These experiments have demonstrated that the administration of less than 9 mg of propionamide and/or butyramide to H. pylori cultures, in loads recognized to cause infection (106–109 cells), resulted in the formation of detectable and/or quantifiable amounts of propionic and/or butyric acids after 30 min incubation. As such, propionic and butyric acids can be used as biomarkers for H. pylori upon incubation with the corresponding amides. SPME–GC–qMS was also used to verify the hepatic stability of the acids. These experiments were conducted in mouse liver cells and revealed no signs of metabolization that could compromise their bioavailability in future in vivo assays. Moreover, SPME–GC–qMS permitted the detection of both acids in amounts as low as 0.8 μg in systems mimicking exhaled breath, demonstrating the sensitivity of the method for these compounds. Figure HS-SPME-GC-qMS (m/z 73, 74 and 88) chromatograms of the vapour phase resulting from the conversion of butyramide and propionamide into butyric and propionic acids by H. pylori.
Keywords: Helicobacter pylori ; H. pylori biomarker; H. pylori detection; Aliphatic amidase; Amide

Late diagnosis of hepatocarcinoma (HCC) is one of the most primary factors for the poor survival of patients. Thereby, identification of sensitive and specific biomarkers for HCC early diagnosis is of great importance in biological medicine to date. In the present study, serum metabolites of the HCC patients and healthy controls were investigated using the improved liquid chromatography–mass spectrometry (LC/MS). A wavelet-based method was utilized to find and align peaks of LC–MS. The characteristic peaks were selected by performing a two-sample t test statistics (p value <0.05). Clustering analysis based on principal component analysis showed a clear separation between HCC patients and healthy individuals. The serum metabolite, namely 1-methyladenosine, was identified as the characteristic metabolite for HCC. Moreover, receiver–operator curves were calculated with 1-methyladenosine and/or alpha fetal protein (AFP). The higher area under curve value was achieved in 1-methyladenosine group than AFP group (0.802 vs. 0.592), and the diagnostic model combining 1-methyladenosine with AFP exhibited significant improved sensitivity, which could identify those patients who missed the diagnosis of HCC by determining serum AFP alone. Overall, these results suggested that LC/MS-based metabonomic study is a potent and promising strategy for identifying novel biomarkers of HCC.
Keywords: Biomarker; Liquid chromatography–mass spectrometry; Metabonomics; Hepatocarcinoma

1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene (TMBB-Su), a new BODIPY-based fluorescent probe, was designed and synthesized for the labeling of amino compounds. It was used as a pre-column derivatizing reagent for determination of amino acid neurotransmitters by high-performance liquid chromatography (HPLC). The fluorescence quantum yield in acetonitrile increased from 0.84 to 0.95 when it reacted with amino acid neurotransmitters. Derivatization of TMBB-Su with seven amino acid neurotransmitters was completed within 30 min at 25 °C in 24.0 mmol L−1 pH 7.8 boric acid buffer. The separation was performed on a C18 column with methanol–water–buffer 55:35:10 (v/v) as mobile phase (buffer: 0.10 mol L−1 H3Cit–0.10 mol L−1 NaOH). Interference from the other concomitant amino acids was eliminated successfully by means of pH gradient elution. With fluorescence detection at 494 and 504 nm for excitation and emission, respectively, the limits of detection (signal-to-noise ratio = 3) were from 2.1 to 12.0 nmol L−1. The proposed method has been used to determine amino acid neurotransmitters in the cerebral cortex of mice with cerebral ischemia at the convalescence stage with satisfactory recoveries varying from 94.9 to 105.2%.
Keywords: 1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene; Amino acid neurotransmitter; HPLC; Fluorescence detection

Phenytoin (PHT), phenobarbital (PHB), lamotrigine (LTG), and topiramate (TPM) are some of the most widely used antiepileptic drugs (AEDs). Monitoring of their concentrations in serum is important for the treatment of epilepsy. A reference measurement procedure (RMP) for certification of PHT, PHB, LTG, and TPM in serum has been developed and critically evaluated. Isotopically labeled compounds of PHT, PHB, LTG, and TPM are used as internal standards for the four AEDs. The four drugs and their respective labeled internal standards are simultaneously extracted from serum using solid-phase extraction prior to reversed-phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a C18 column. Electrospray ionization (ESI) in the positive ion mode for PHT and LTG, and in the negative ion mode for PHB and TPM were used. The recovery of AEDs added to serum (accuracy of the extraction method) was evaluated by recovery studies of measuring the four drugs in spiked samples with known drug levels. The recoveries of the added drugs ranged from 98.6% to 102.0%. The absolute recoveries (extraction efficiencies) of the four drugs with this method ranged from 97% to 100%. Excellent repeatability was obtained for the four drugs with between-set coefficients of variation (CVs) within 1%. The type B components estimates are conservatively large and are considerably larger than the type A component. Therefore, we use the usual metrological expansion factor of 2 to provide an approximate 95% coverage interval. The relative expanded uncertainties for the four AEDs ranged from 2.3% to 2.4%. This LC-MS/MS RMP for PHT, PHB, LTG, and TPM in serum demonstrating good accuracy and precision can be used to assess the accuracy of routine methods used in clinical laboratories. Figure
Keywords: Antiepileptic drugs; Reference measurement procedure; Electrospray ionization; Isotope dilution; Liquid chromatography/tandem mass spectrometry; Solid-phase extraction

Microanalysis of the antiretroviral nevirapine in human hair from HIV-infected patients by liquid chromatography-tandem mass spectrometry by Yong Huang; Qiyun Yang; Kwangchae Yoon; Yvonne Lei; Robert Shi; Winnie Gee; Emil T. Lin; Ruth M. Greenblatt; Monica Gandhi (1923-1933).
Sufficient drug exposure is crucial for maintaining durable responses to HIV treatments. However, monitoring drug exposure using single blood samples only provides short-term information and is highly subject to intra-individual pharmacokinetic variation. Drugs can accumulate in hair over a long period of time, so hair drug levels can provide drug exposure information over prolonged periods. We now report on a specific, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method for measuring nevirapine (NVP), a widely used antiretroviral drug, levels in human hair using even a single short strand of hair. Hair samples are cut into small segments, and the drug is extracted in methanol/trifluoroacetic acid (v/v, 9:1) shaken at 37 °C in a water bath overnight, followed by liquid–liquid extraction under alkaline conditions. The extracted samples are then separated on a BDS-C18 column with a mobile phase composed of 50% acetonitrile containing 0.15% acetic acid and 4 mM ammonium acetate with an isocratic elution for a total run time of 3 min and detected by triple quadrupole electrospray multiple reaction mode at precursor/product ion at 267.0 > 225.9 m/z. Deuterated nevirapine-d5 was used as an internal standard. This method was validated from 0.25 to 100 ng/mg using 2 mg hair samples. The accuracies for spiked NVP hair control samples were 98–106% with coefficients of variation (CV) less than 10%. The CV for incurred hair control samples was less than 7%. The extraction efficiency for incurred control hair samples was estimated at more than 95% by repeated extractions. This method has been successfully applied to analyze more than 1,000 hair samples from participants in a large ongoing cohort study of HIV-infected participants. We also showed that NVP in human hair can easily be detected in a single short strand of hair. This method will allow us to identify drug non-adherence using even a single strand of hair. Figure Therapeutic drug monitoring using hair
Keywords: Antiretroviral drug; Nevirapine; Hair; LC-MS/MS; TDM; Adherence

The usefulness of ion mobility spectrometry as a screening methodology for the on-site benzodiazepine analysis in saliva samples has been critically evaluated. The procedure involved the injection of clear supernatant extracts after centrifugation and provided limit of detection values ranging from 2.0 to 18 μg L−1, and a precision, expressed as relative standard deviation, from 2.9% to 16%, depending on the different benzodiazepines studied. Those values are appropriate for their positive identification in saliva samples in which benzodiazepine concentration, after a chronic or acute dose, is in the range of 2–30 μg L−1. Problems related with overlapped benzodiazepine signals have been successfully overcome by application of multivariate curve resolution, which is a helpful tool to improve the resolution of the technique, without sacrificing the method simplicity and frequency of analysis. The possibility of false positives caused by the presence of interferents with the same drift time as the benzodiazepines and the possibility of false negatives due to the presence of interferents by competitive ionization have been critically evaluated. The satisfactory results obtained for the analysis of real saliva samples after an acute dose of diazepam through sublingual and oral intakes confirm the capability of the technique to be used as a screening methodology in the analysis of benzodiazepines in oral fluids.
Keywords: Ion mobility spectrometry; Benzodiazepines; Drug testing; Saliva; Oral fluid

Data quality in tissue analysis using desorption electrospray ionization by Allison L. Dill; Livia S. Eberlin; Anthony B. Costa; Demian R. Ifa; R. Graham Cooks (1949-1961).
There has been a recent surge in applications of mass spectrometry (MS) to tissue analysis, particularly lipid-based tissue imaging using ambient ionization techniques. This recent growth highlights the need to examine the effects of sample handling, storage conditions, and experimental protocols on the quality of the data obtained. Variables such as time before freezing after organ removal, storage time at −80 °C, time stored at room temperature, heating, and freeze/thaw cycles were investigated for their effect on the data quality obtained in desorption electrospray ionization (DESI)-MS using mouse brain. In addition, analytical variables such as tissue thickness, drying times, and instrumental conditions were also examined for their impact on DESI-MS data. While no immediate changes were noted in the DESI-MS lipid profiles of the mouse brain tissue after spending 1 h at room temperature when compared to being frozen immediately following removal, minor changes were noted between the tissue samples after 7 months of storage at −80 °C. In tissue sections stored at room temperature, degradation was noted in 24 h by the appearance of fatty acid dimers, which are indicative of high fatty acid concentrations, while in contrast, those sections stored at −80 °C for 7 months showed no significant degradation. Tissue sections were also subjected to up to six freeze/thaw cycles and showed increasing degradation following each cycle. In addition, tissue pieces were subjected to 50 °C temperatures and analyzed at specific time points. In as little as 2 h, degradation was observed in the form of increased fatty acid dimer formation, indicating that enzymatic processes forming free fatty acids were still active in the tissue. We have associated these dimers with high concentrations of free fatty acids present in the tissue during DESI-MS experiments. Analytical variables such as tissue thickness and time left to dry under nitrogen were also investigated, with no change in the resulting profiles at thickness from 10 to 25 μm and with optimal signal obtained after just 20 min in the dessicator. Experimental conditions such as source parameters, spray solvents, and sample surfaces are all shown to impact the quality of the data. Inter-section (relative standard deviation (%RSD), 0.44–7.2%) and intra-sample (%RSD, 4.0–8.0%) reproducibility data show the high quality information DESI-MS provides. Overall, the many variables investigated here showed DESI-MS to be a robust technique, with sample storage conditions having the most effect on the data obtained, and with unacceptable sample degradation occurring during room temperature storage. Figure Recent growth in tissue analysis by mass spectrometry highlights the need to control sample handling, storage conditions and experimental protocols to optimize data quality.
Keywords: Ambient ionization; Desorption electrospray ionization; Imaging mass spectrometry; Lipids; Molecular imaging; Reproducibility

Surface-assisted laser desorption/ionization-mass spectrometry using TiO2-coated steel targets for the analysis of small molecules by Harald Sonderegger; Christoph Rameshan; Harald Lorenz; Frederik Klauser; Mariska Klerks; Matthias Rainer; Rania Bakry; Christian W. Huck; Günther K. Bonn (1963-1974).
Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI–MS) measurements in the low-molecular-mass region, ranging from 0 to 1000 Daltons are very often difficult to perform because of signal interferences originating from matrix ions. In order to overcome this problem, a stainless steel target was coated with a homogeneous titanium dioxide layer. The layer obtained was further investigated for its ability to desorb small molecules, e.g., amino acids, sugars, poly(ethylene glycol) (PEG) 200, or extracts from Cynara scolymus leaves. The stability of the layer was determined by repeated measurements on the same target location, which was monitored by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) before and after surface-assisted laser desorption/ionization (SALDI) analysis. In addition, this titanium dioxide layer was compared with an already published method with titanium dioxide nanopowder as inorganic matrix. As a result of this work, the titanium dioxide layer produced minimal background interference, enabling simple interpretation of the detected mass spectra. Furthermore, the TiO2 coating provides a target that can be reused many times for SALDI–MS measurements.
Keywords: MALDI; LDI–MS; SALDI; TiO2 coating; Metabolites

One of the features of protein-based stable isotope probing is the parallel identification of differentially labeled peptide forms and the accurate calculation of their relative isotope abundances. The level of incorporation is informative of the metabolic activity of the species that synthesized the said protein and peptide. To model the carbon flux in a microbial community, an accurate assessment is crucial. Since the initial processes in carbon consumption are one of the most interesting objectives in microbial ecology, the methodology to detect low amounts of incorporation was tuned, and the limits of robust detection were analyzed. For this, Pseudomonas fluorescens DSM 50090T was grown on galactose using different ratios of 12C/13C galactose from 10% down to 0.1% labeled galactose. After prolonged cultivation to ensure complete labeling, protein samples were separated by one-dimensional gel electrophoresis, subsequently tryptically digested and analyzed by ultra-performance liquid chromatography (UPLC) Orbitrap tandem mass spectrometry (MS/MS) measurements. The isotopic patterns from identified peptides in the mass spectra were used to calculate the 13C relative isotope abundance (RIA) in the respective peptides. The statistic distribution of the RIA values in dependence of the number of analyzed peptides was compared between the different ratios of unlabeled/labeled substrate. The acquired data showed that the applied method is capable of detecting a difference in 13C incorporation of ±0.1% RIA based on at least 20 peptides. This sensitivity makes protein–stable isotope probing a valuable method for quantitative assessment of species specific metabolic activity in metaproteomics.
Keywords: Stable isotope probing; Protein-SIP; Mass spectrometry; Relative isotope abundance; 13C incorporation

Neutral loss and precursor ion scan tandem mass spectrometry for study of activated benzopyrene–DNA adducts by D. Compagnone; R. Curini; G. D’Ascenzo; M. Del Carlo; C. Montesano; S. Napoletano; M. Sergi (1983-1991).
Methodology for detection of activated benzo[a]pyrene (B[a]P)–nucleoside adducts by liquid chromatography–tandem mass spectrometry is reported. Adducts of B[a]P-dihydrodiol epoxide (B[a]PDE) with guanosine and adenosine have been detected for the first time by use of precursor ion scan and neutral loss scan. B[a]P was then activated by use of UV irradiation and some of the products obtained have been identified by taking advantage of the information obtained for B[a]PDE. Photoactivation has also been carried out in the presence of hydrogen peroxide; this resulted in a higher yield of products with increased production of BaP diones. The reactivity of these compounds toward nucleosides has been tested. The proposed method was successfully used for detection of one stable guanosine–B[a]P dione adduct. Figure Interactions between activated B[a]P and DNA; MS/MS detection strategies
Keywords: Benzopyrene; DNA adducts; Neutral loss scan; Precursor ion scan; Photoactivation; PAHs

Measuring silver nanoparticle dissolution in complex biological and environmental matrices using UV–visible absorbance by Justin M. Zook; Stephen E. Long; Danielle Cleveland; Carly Lay A. Geronimo; Robert I. MacCuspie (1993-2002).
Distinguishing the toxic effects of nanoparticles (NPs) themselves from the well-studied toxic effects of their ions is a critical but challenging measurement for nanotoxicity studies and regulation. This measurement is especially difficult for silver NPs (AgNPs) because in many relevant biological and environmental solutions, dissolved silver forms AgCl NPs or microparticles. Simulations predict that solid AgCl particles form at silver concentrations greater than 0.18 and 0.58 μg/mL in cell culture media and moderately hard reconstituted water (MHRW), respectively. The AgCl NPs are usually not easily separable from AgNPs. Therefore, common existing total silver techniques applied to measure AgNP dissolution, such as inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption, cannot accurately measure the amount of silver remaining in AgNP form, as they cannot distinguish Ag oxidation states. In this work, we introduce a simple localized surface plasmon resonance (LSPR) UV–visible absorbance measurement as a technique to measure the amount of silver remaining in AgNP form for AgNPs with constant agglomeration states. Unlike other existing methods, this absorbance method can be used to measure the amount of silver remaining in AgNP form even in biological and environmental solutions containing chloride because AgCl NPs do not have an associated LSPR absorbance. In addition, no separation step is required to measure the dissolution of the AgNPs. After using ICP-MS to show that the area under the absorbance curve is an accurate measure of silver in AgNP state for unagglomerating AgNPs in non-chloride-containing media, the absorbance is used to measure dissolution rates of AgNPs with different polymer coatings in biological and environmental solutions. We find that the dissolution rate decreases at high AgNP concentrations, 5 kDa polyethylene glycol thiol coatings increase the dissolution rate, and the rate is much higher in cell culture media than in MHRW. Figure
Keywords: Silver colloid; Dissolution; Silver ion; ICP-MS; Localized surface plasmon resonance absorbance

Increasing the sensitivity in DNA microarray hybridization can significantly enhance the capability of microarray technology for a wide range of research and clinical diagnostic applications, especially for those with limited sample biomass. To address this issue, using reverse microemulsion method and surface chemistry, a novel class of homogenous, photostable, highly fluorescent streptavidin-functionalized silica nanoparticles was developed, in which Alexa Fluor 647 (AF647) molecules were covalently embedded. The coating of bovine serum albumin on the resultant fluorescent particles can greatly eliminate nonspecific background signal interference. The thus-synthesized fluorescent nanoparticles can specifically recognize biotin-labeled target DNA hybridized to the microarray via streptavidin–biotin interaction. The response of this DNA microarray technology exhibited a linear range within 0.2 to 10 pM complementary DNA and limit of detection of 0.1 pM, enhancing microarray hybridization sensitivity over tenfold. This promising technology may be potentially applied to other binding events such as specific interactions between proteins. Figure
Keywords: DNA microarray; Sensitivity; Silica nanoparticles chemically doped dye; Biotin–streptavidin interaction; Reverse microemulsion reaction

Benzylpiperazine (BZP) is an amphetamine-type stimulant, which was legally available in New Zealand and widely used in “Party Pills” until reclassification as a Class C drug in April 2008. BZP was included as part of a multi-analyte method developed for hair screening using high-performance liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS). A 20-mg sample of hair is extracted and partially purified using mixed-mode solid-phase extraction cartridges prior to analysis by LC-MS/MS. The method was developed as a broad screen for drugs of abuse (including amphetamines, opiates, and benzodiazepines), with only the BZP results being presented here. The assay was validated and found to be linear over the range of 0.085 to 8.65 ng/mg with correlation coefficient of r 2 ≥ 0.99. Blank hair samples spiked with BZP at 0.22 and 2.16 ng/mg gave intra- and inter-day precision coefficients of variation of ≤10% (n = 6 per day, 3 days) at both levels and calculated extraction efficiencies of 78% and 91%, respectively. The results from the samples submitted to the laboratory for BZP analysis showed 11% were positive (n = 126). The mean BZP level was 3.9 ng/mg (range, 0.4–33 ng/mg; the result was extrapolated when above the calibration). These data are the first available showing the levels expected from users of BZP. Figure Distribution of BZP in hair (ng/mg) from 14 positive hair samples, using LC-MS/MS detection
Keywords: Hair; LC-MS/MS; Drugs of abuse; 1-Benzylpiperazine; BZP

A new procedure for extraction of collagen from modern and archaeological bones for 14C dating by F. Maspero; S. Sala; M. E. Fedi; M. Martini; A. Papagni (2019-2023).
Bones are potentially the best age indicators in a stratigraphic study, because they are closely related to the layer in which they are found. Collagen is the most suitable fraction and is the material normally used in radiocarbon dating. Bone contaminants can strongly alter the carbon isotopic fraction values of the samples, so chemical pretreatment for 14 C dating by accelerator mass spectrometry (AMS) is essential. The most widespread method for collagen extraction is based on the Longin procedure, which consists in HCl demineralization to dissolve the inorganic phase of the samples, followed by dissolution of collagen in a weak acid solution. In this work the possible side effects of this procedure on a modern bone are presented; the extracted collagen was analyzed by ATR-IR spectroscopy. An alternative procedure, based on use of HF instead of HCl, to minimize unwanted degradation of the organic fraction, is also given. A study by ATR-IR spectroscopic analysis of collagen collected after different demineralization times and with different acid volumes, and a study of an archaeological sample, are also presented.
Keywords: Radiocarbon; Archaeometry; Biological samples; Bones; Collagen

Impact of pump flow fluctuations on post column online ID-ICP-MS by Claudia Swart; Olaf Rienitz; Detlef Schiel (2025-2031).
In post column online isotope dilution mass spectrometry (IDMS), the stability of the spike mass flow is a key element. Changes in viscosity or fluctuations in the pump rate of the peristaltic pump may affect the results of post column online IDMS measurements. It was shown by simulating random fluctuations and studying the changes in the resulting integrals of the isotope ratio chromatogram of the sample that even small fluctuations, observable when using peristaltic pumps, can influence the result and especially its uncertainty. The use of a balance to continuously monitor the mass flow of the spike during the measurement which we presented in a previous publication allows now to correct the isotope ratio chromatogram for these fluctuations. Subsequently, the simulated effect was verified experimentally for the determination of Se-Met in the human serum reference material BCR 637, where the corrected mass fraction was plainly closer to the mass fraction obtained by species specific IDMS. Additional attention was paid to the fact that there is a time shift between the observation of the fluctuations in the pump rate and the detection of these fluctuations in the ICP-MS. Figure The success of correcting for pump flow fluctuations depends strongly on the application of the accurate time shift. Green to red: increasing deviation from the accurate time shift results in an increasing ineffectiveness of the correction applied
Keywords: Online double IDMS; Mass flow fluctuations; Species analysis; Se-methionine; Human serum