Analytica Chimica Acta (v.810, #C)

Estimation of the limit of detection using information theory measures by Jordi Fonollosa; Alexander Vergara; Ramón Huerta; Santiago Marco (1-9).
Definitions of the limit of detection (LOD) based on the probability of false positive and/or false negative errors have been proposed over the past years. Although such definitions are straightforward and valid for any kind of analytical system, proposed methodologies to estimate the LOD are usually simplified to signals with Gaussian noise. Additionally, there is a general misconception that two systems with the same LOD provide the same amount of information on the source regardless of the prior probability of presenting a blank/analyte sample. Based upon an analogy between an analytical system and a binary communication channel, in this paper we show that the amount of information that can be extracted from an analytical system depends on the probability of presenting the two different possible states. We propose a new definition of LOD utilizing information theory tools that deals with noise of any kind and allows the introduction of prior knowledge easily. Unlike most traditional LOD estimation approaches, the proposed definition is based on the amount of information that the chemical instrumentation system provides on the chemical information source. Our findings indicate that the benchmark of analytical systems based on the ability to provide information about the presence/absence of the analyte (our proposed approach) is a more general and proper framework, while converging to the usual values when dealing with Gaussian noise.
Keywords: Limit of detection; Information theory; Mutual information; Heteroscedasticity; False positive/negative errors; Gas discrimination and quantification;

Heavy metal ion pollution poses severe risks in human health and environmental pollutant, because of the likelihood of bioaccumulation and toxicity. Driven by the requirement to monitor trace-level mercury ion (Hg2+), herein we construct a new DNA-based sensor for sensitive electrochemical monitoring of Hg2+ by coupling target-induced formation of gold amalgamation on DNA-based sensing platform with gold amalgamation-catalyzed cycling signal amplification strategy. The sensor was simply prepared by covalent conjugation of aminated poly-T(25) oligonucleotide onto the glassy carbon electrode by typical carbodiimide coupling. Upon introduction of target analyte, Hg2+ ion was intercalated into the DNA polyion complex membrane based on T–Hg2+–T coordination chemistry. The chelated Hg2+ ion could induce the formation of gold amalgamation, which could catalyze the p-nitrophenol with the aid of NaBH4 and Ru(NH3)6 3+ for cycling signal amplification. Experimental results indicated that the electronic signal of our system increased with the increasing Hg2+ level in the sample, and has a detection limit of 0.02 nM with a dynamic range of up to 1000 nM Hg2+. The strategy afforded exquisite selectivity for Hg2+ against other environmentally related metal ions. In addition, the methodology was evaluated for the analysis of Hg2+ in spiked tap-water samples, and the recovery was 87.9–113.8%.
Keywords: DNA-based sensor; Gold amalgamation; Electrochemistry; Mercury ion; Cycling signal amplification;

The work aims at developing a simple and rapid method for the quantification of dopamine (DA), serotonin (5-HT) and norepinephrine (NE) in human urine. The urinary levels of these biogenic amines can be correlated with several pathological conditions concerning heart disease, stress, neurological disorders and cancerous tumors. The proposed analytical approach is based on the use of solid phase microextraction (SPME) combined with gas chromatography–triple quadrupole mass spectrometry (GC–QqQ-MS) after a fast derivatization of both aliphatic amino and phenolic moieties by propyl chloroformate. The variables influencing the derivatization reaction were reliably optimized by the multivariate approach of “Experimental design”. The optimal conditions were obtained by performing derivatization with 100 μL of propyl chloroformate and 100 μL of pyridine. The extraction ability of five commercially available SPME fibers was evaluated in univariate mode and the best results were obtained using the polyacrylate fiber. The variables affecting the efficiency of SPME analysis were again optimized by the multivariate approach of “Experimental design” and, in particular, a central composite design (CCD) was applied. The optimal values were extraction in 45 min at room temperature, desorption temperature at 300 °C, no addition of NaCl. Assay of derivatized analytes was performed by using a gas chromatography–triple quadrupole mass spectrometry (GC–QqQ-MS) system in selected reaction monitoring (SRM) acquisition. An evaluation of all analytical parameters demonstrates that the developed method provides satisfactory results. Indeed, very good linearities were achieved in the tested calibration range with correlation coefficient values of 0.9995, 0.9999 and 0.9997 for DA, 5-HT and NE, respectively. Accuracies and RSDs calculated for between-run and tested at concentrations of 30, 200, and 800 μg L−1 were in the range from 92.8% to 103.0%, and from 0.67 to 4.5%, respectively. Finally, the LOD values obtained can be considered very good (0.587, 0.381 and 1.23 μg L−1 for DA, 5-HT and NE, respectively).
Keywords: Dopamine; Serotonin; Norepinephrine; Catecholamines; Gas chromatography; Solid phase microextraction;

A sensitive, economical, and miniaturized self-assembly pipette tip graphene solid-phase extraction (PT-G-SPE) coupled with liquid chromatography fluorescence detection (LC-FD) was developed for rapid extraction and determination of three sulfonamide antibiotics (SAs) in environmental water samples. The PT-G-SPE cartridge, assembled by packing 1.0 mg of graphene as sorbent into a 100 μL pipette tip, showed high adsorption capacity for the SAs owing to the large surface area and unique structure of graphene. The factors that affected the extraction efficiency of PT-G-SPE, including sample volume, pH, sorbent amount, washing solvent and eluent solvent were optimized. Good linearity for SAs was obtained in a range of 2–4000 pg mL−1 with correlation coefficients (r 2) ≥ 0.9993. The recoveries of the SAs at three spiked levels ranged from 90.4% to 108.2% with relative standard deviations (RSD) ≤ 6.3%. In comparison with other sorbents such as C18, HLB, SCX, PCX, and multiwalled carbon nanotubes, one advantage of using graphene as sorbent of pipette tip solid-phase extraction (PT-SPE) was that PT-G-SPE could adsorb larger sample volume (10 mL) at a small amount of sorbent (1 mg) and low solvent consumption with good extraction efficiency, which not only increased the fraction of analytes to LC and the sensitivity of SAs determination, but also reduced the cost and pollution.
Keywords: Pipette tip graphene solid-phase extraction; Sulfonamide antibiotics; Liquid chromatography-fluorescence detection; Environmental water;

In this article, we demonstrate a novel approach to enhancing the sensitivity of enzyme-linked immunosorbent assays (ELISA) through pre-concentration of the enzyme reaction product (resorufin/4-methylumbelliferone) in free solution. The reported pre-concentration was accomplished by transporting the resorufin/4-methylumbelliferone molecules produced in the ELISA process towards a high ionic-strength buffer stream in a microfluidic channel while applying a voltage drop across this merging region. A sharp change in the electric field around the junction of the two liquid streams was observed to abruptly slow down the negatively charged resorufin/4-methylumbelliferone species leading to the reported pre-concentration effect based on the field amplified stacking (FAS) technique. It has been shown that the resulting enhancement in the detectability of the enzyme reaction product significantly improves the signal-to-noise ratio in the system thereby reducing the smallest detectable analyte concentration in the ELISA method. Applying the above-described approach, we were able to detect mouse anti-BSA and human TNF-α at concentrations nearly 60-fold smaller than that possible on commercial microwell plates. For the human TNF-α sample, this improvement in assay sensitivity corresponded to a limit of detection (LOD) of 0.102 pg mL−1 using the FAS based microfluidic ELISA method as compared to 7.03 pg mL−1 obtained with the traditional microwell plate based approach. Moreover, because our ELISAs were performed in micrometer sized channels, they required sample volumes about two orders of magnitude smaller than that consumed in the latter case (1 μL versus 100 μL).
Keywords: Enzyme-linked immunosorbent assay; Field amplified stacking; Microfluidic; Pre-concentration; TNF-α;

Varlet et al. recently proposed a headspace-gas chromatography–mass spectrometry (HS-GC–MS) method applicable for the routine determination of CO2 in gaseous biologic matrices. This developed bioanalytical method was fully validated according to the SFSTP 1997 guidelines using the accuracy profile as a graphical decision-making tool.In this letter, we discuss the validity of HS-GC–MS method based on the newest SFSTP guideline. In particular, we demonstrate by the estimation of the β-expectation tolerance interval that the error total exceeds the acceptance limits (30%) for the second concentration level (0.5 μmol mL−1 vial HS).Furthermore, we show through the risk profile that the probability to have future results inside the ±30% acceptance limits is smaller than 95%.
Keywords: Analytical validation; SFSTP 2003; SFSTP 1997; Accuracy profile; Tolerance interval;

Saffaj et al. recently criticized our method of monitoring carbon dioxide in human postmortem cardiac gas samples using Headspace-Gas Chromatography–Mass Spectrometry.According to the authors, their demonstration, based on the latest SFSTP guidelines (established after 2007 [1,2]) fitted for the validation of drug monitoring bioanalytical methods, has put in evidence potential errors.However, our validation approach was built using SFSTP guidelines established before 2007 [3–6]. We justify the use of these guidelines because of the post-mortem context of the study (and not clinical) and the gaseous state of the sample (and not solid or liquid). Using these guidelines, our validation remains correct.
Keywords: Carbon dioxide; Headspace gas extraction; Accuracy profile; Gaseous biological matrices;

In this study, an ultra-high performance liquid chromatography–quadrupole-orbital ion trap mass spectrometry (UHPLC–Q-orbitrap MS) method was developed and validated for simultaneous determination of 96 pharmaceuticals, plant toxins, and other plant secondary metabolites in herbal dietary supplements. Target analytes were extracted from samples using the QuEChERS (quick easy cheap effective rugged safe) procedure. The instrument was operated in full MS–data dependent tandem mass spectrometry (full MS–dd-MS/MS) acquisition mode which enabled collection of quantitative high resolution (HR) full mass spectral data and confirmatory HR MS/MS data in a single run. The method provided excellent selectivity in both full MS and dd-MS/MS mode. Under optimized collision energy settings, product ion spectra containing both precursor and two or more product ions were obtained for most of the analytes. Limits of detection (LODs) and limits of quantification (LOQs) for the method differed significantly for the examined matrices. LODs ≤ 10 μg kg−1 and LOQs ≤ 50 μg kg−1 were obtained for 48 to 81% of target compounds across five different matrices. With the exception of highly polar analytes, the optimized QuEChERS extraction procedure provided acceptable recoveries in the range 70%–120%. The precision of the method, characterized as the relative standard deviation (RSD, n  = 5), was ≤25% and ≤18% at spiking concentrations of 50 μg kg−1 and 500 μg kg−1, respectively. Because of variations in matrix effects in extracts of herbal dietary supplements that differed in composition, the method of standard additions and an approach based on dilution of matrix components followed by quantification using solvent standards were applied for quantification. The procedure was used to examine commercial dietary supplements for the 96 analytes of interest. To the best of our knowledge, this is the first report of an integrated analysis and quantification of this wide range of compounds.
Keywords: Dietary supplements; Pharmaceuticals; Plant toxins; Ultra-high performance liquid chromatography; Quadrupole-orbitrap mass spectrometer;

While the occurrence of pharmaceuticals in the aquatic environment has been extensively investigated, their environmental fate is less thoroughly explored. Scarce information on their transformation pathways and transformation products (TPs) limits conventional target analytical approaches. In this study, samples from water/sediment tests were analyzed by ultrahigh performance liquid chromatography interfaced with quadrupole time-of-flight mass spectrometry (UHPLC/QToF-MS). A data processing method based on peak detection, time-trend filtration and structure assignment was established to provide an efficient way for identifying the key TPs in terms of persistence; all software used for the individual steps of this method is freely available. The accurate mass and meaningful time-trends were major contributors in facilitating the isolation of plausible TP peaks. In total, 16 TPs from 9 parent pharmaceuticals were identified. Eleven out of the 16 TPs were confirmed by corresponding reference standards; no standards were available for the remaining TPs. For additional 6 potential TPs, a molecular formula was suggested but no additional structural information could be generated. Among the TPs identified in the water/sediment tests, carbamazepine-10,11-epoxide (parent: carbamazepine), saluamine (parent: furosemide), chlorothiazide and 4-amino-6-chloro-1,3-benzenedisulfonamide (parent of both: hydrochlorothiazide), and 1-naphthol (parent: propranolol) accumulated over the entire incubation period of 35 days.
Keywords: Pharmaceuticals; Transformation products; Water/sediment test; Ultrahigh performance liquid chromatography/high resolution mass spectrometry; In silico data processing;

Fluorescent blood glucose monitor by hemin-functionalized graphene quantum dots based sensing system by Yuezhen He; Xiaoxun Wang; Jian Sun; Shoufeng Jiao; Hongqi Chen; Feng Gao; Lun Wang (71-78).
In the present work, a highly sensitive and specific fluorescent biosensor for blood glucose monitoring is developed based on hemin-functionalized graphene quantum dots (GQDs) and glucose oxidase (GOx) system. The GQDs which are simply prepared by pyrolyzing citric acid exhibit strong fluorescence and good water-solubility. Due to the noncovalent assembly between hemin and GQDs, the addition of hemin can make hydrogen peroxide (H2O2) to destroy the passivated surface of GQDs, leading to significant fluorescence quenching of GQDs. Based on this effect, a novel fluorescent platform is proposed for the sensing of glucose. Under the optimized conditions, the linear range of glucose is from 9 to 300 μM, and the limit of detection is 0.1 μM. As unique properties of GQDs, the proposed biosensor is green, simple, cost-efficient, and it is successfully applied to the determination of glucose in human serum. In addition, the proposed method provides a new pathway to further design the biosensors based on the assembly of GQDs with hemin for detection of biomolecules.
Keywords: Graphene quantum dots; Hemin; Hydrogen peroxide; Glucose; Glucose oxidase; Biosensor;

Here we report the characterization of an electrochemical mercury (Hg2+) sensor constructed with a methylene blue (MB)-modified and thymine-containing linear DNA probe. Similar to the linear probe electrochemical DNA sensor, the resultant sensor behaved as a “signal-off” sensor in alternating current voltammetry and cyclic voltammetry. However, depending on the applied frequency or pulse width, the sensor can behave as either a “signal-off” or “signal-on” sensor in square wave voltammetry (SWV) and differential pulse voltammetry (DPV). In SWV, the sensor showed “signal-on” behavior at low frequencies and “signal-off” behavior at high frequencies. In DPV, the sensor showed “signal-off” behavior at short pulse widths and “signal-on” behavior at long pulse widths. Independent of the sensor interrogation technique, the limit of detection was found to be 10 nM, with a linear dynamic range between 10 nM and 500 nM. In addition, the sensor responded to Hg2+ rather rapidly; majority of the signal change occurred in <20 min. Overall, the sensor retains all the characteristics of this class of sensors; it is reagentless, reusable, sensitive, specific and selective. This study also highlights the feasibility of using a MB-modified probe for real-time sensing of Hg2+, which has not been previously reported. More importantly, the observed “switching” behavior in SWV and DPV is potentially generalizable and should be applicable to most sensors in this class of dynamics-based electrochemical biosensors.
Keywords: Alternating current voltammetry; Cyclic voltammetry; Square wave voltammetry; Differential pulse voltammetry; Methylene blue; Thymine–mercury(II)–thymine;

The influence of film morphology and illumination conditions on the sensitivity of porphyrins-coated ZnO nanorods by Gabriele Magna; Yuvaraj Sivalingam; Eugenio Martinelli; Giuseppe Pomarico; Francesco Basoli; Roberto Paolesse; Corrado Di Natale (86-93).
ZnO and porphyrins have complementary properties that make their combination attractive for diverse applications such as photovoltaic and chemical sensing. Among the other features, the organic layer morphology is supposed to influence both the chemical sensitivity and the charge transfer processes. In this paper, we studied the influence of the film morphology on the sensing properties by comparing porphyrins coated ZnO nanorods obtained with two different methods. In the first approach, each porphyrin unit is grafted onto preformed ZnO nanorods by a carboxylic group as linker. The second method is a one-pot procedure, where ZnO nanorods growth occurs in the presence of the water soluble tetrakis-(4-sulfonatophenyl)porphyrin. In both cases the macrocycles share the same Zn-tetraphenylporphyrin core structure, but decorated with different peripheral groups, necessary to comply with the material growth conditions.The adsorption of volatile organic molecules has been monitored measuring the contact potential difference between the sensitive surface and a gold electrode, by means of a Kelvin probe setup. Sensitive signals have been measured both in dark and under visible light. The results show that material preparation affects both the sensitivities to gases and light. A chemometric analysis of four sensors (first and second growth method, measured in dark and in light) shows two main evidences: (a) the interaction between volatile compounds and the sensing layer is largely dominated by non-specific dispersion interaction and (b) the signal of the four sensors becomes rather uncorrelated when the contribution of the dispersion interaction is removed. These results indicate that the differences due to film morphology are enough to differentiate the sensor behaviour, even when the same porphyrin nucleus is used as sensing element. This feature provides an additional degree of freedom for the development of gas sensor arrays.
Keywords: ZnO nanorods; Porphyrins; Volatile organic compounds; Kelvin probe;

A PrinCE autosampler was coupled to a Q-Exactive mass spectrometer by an electrokinetically pumped sheath–flow nanospray interface to perform automated capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). 20 ng aliquots of an Escherichia coli digest were injected to evaluate the system. Eight sequential injections over an 8-h period identified 1115 ± 70 (relative standard deviation, RSD = 6%) peptides and 270 ± 8 (RSD = 3%) proteins per run. The average RSDs of migration time, peak intensity, and peak area were 3%, 24% and 19%, respectively, for 340 peptides with high intensity. This is the first report of an automated CZE-ESI-MS/MS system using the electrokinetically pumped sheath–flow nanospray interface. The results demonstrate that this system is capable of reproducibly identifying over 1000 peptides from an E. coli tryptic digest in a 1-h analysis time.
Keywords: Proteomics; Capillary zone electrophoresis;