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Analytica Chimica Acta (v.760, #)

Editorial Board (pp. iii).

Current trends in separation of plasmid DNA vaccines: A review by Ashraf Ghanem; Robert Healey; Frady G. Adly (pp. 1-15).
Strategies for purifying supercoiled plasmid DNA.Display Omitted► Current trends in the separation and purification of Plasmid DNA vaccines. ► Practical large and analytical scale productions of the plasmid DNA vaccine are discussed. ► Separation challenges. ► New developments and process solutions. ► Future prospective in the field of plasmid DNA vaccines.Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

Keywords: Plasmids; Vaccine; pDNA; Separation; Purification; Chromatography; Monolith


Quantitative analysis of the effect of zidovudine, efavirenz, and ritonavir on insulin aggregation by multivariate curve resolution alternating least squares of infrared spectra by Idoia Martí-Aluja; Itziar Ruisánchez; M. Soledad Larrechi (pp. 16-24).
Display Omitted► The structure of insulin can be changed via interaction with antiretroviral drugs. ► The chemical interaction promotes the formation of aggregates. ► This drug effect was evaluated by MCR-ALS coupled to IR spectroscopy. ► Formation of aggregates was favourable if drugs were able to form hydrogen bonds. ► Higher drug concentrations favoured formation of amorphous aggregates.Quantification of the effect of antiretroviral drugs on the insulin aggregation process is an important area of research due to the serious metabolic diseases observed in AIDS patients after prolonged treatment with these drugs. In this work, multivariate curve resolution alternating least squares (MCR-ALS) was applied to infrared monitoring of the insulin aggregation process in the presence of three antiretroviral drugs to quantify their effect. To evidence concentration dependence in this process, mixtures at two different insulin:drug molar ratios were used. The interaction between insulin and each drug was analysed by1H NMR spectroscopy. In all cases, the aggregation process was monitored during 45min by infrared spectroscopy. The aggregates were further characterised by scanning electron microscopy (SEM). MCR-ALS provided the spectral and concentration profiles of the different insulin–drug conformations that are involved in the process. Their feasible band boundaries were calculated using the MCR-BANDS methodology. The kinetic profiles describe the aggregation pathway and the spectral profiles characterise the conformations involved. The retrieved results show that each of the three drugs modifies insulin conformation in a different way, promoting the formation of aggregates. Ritonavir shows the strongest promotion of aggregation, followed by efavirenz and zidovudine. In the studied concentration range, concentration dependence was only observed for zidovudine, with shorter aggregation time obtained as the amount of zidovudine increased. This factor also affected the aggregation pathway.

Keywords: Insulin aggregation; Zidovudine; Efavirenz; Ritonavir; Multivariate curve resolution alternating least squares; FTIR; 1; H NMR spectroscopy


Sample size planning for classification models by Claudia Beleites; Ute Neugebauer; Thomas Bocklitz; Christoph Krafft; Jürgen Popp (pp. 25-33).
Display Omitted► We compare sample size requirements for classifier training and testing. ► Number of training samples: determine from learning curve. ► Test sample size: specify confidence interval width or model to compare to. ► Classifier testing needs far more samples than training. ► Start with at least 75 cases per class, then refine sample size planning.In biospectroscopy, suitably annotated and statistically independent samples ( e.g. patients, batches, etc.) for classifier training and testing are scarce and costly. Learning curves show the model performance as function of the training sample size and can help to determine the sample size needed to train good classifiers. However, building a good model is actually not enough: the performance must also be proven. We discuss learning curves for typical small sample size situations with 5–25 independent samples per class. Although the classification models achieve acceptable performance, the learning curve can be completely masked by the random testing uncertainty due to the equally limited test sample size. In consequence, we determine test sample sizes necessary to achieve reasonable precision in the validation and find that 75–100 samples will usually be needed to test a good but not perfect classifier. Such a data set will then allow refined sample size planning on the basis of the achieved performance. We also demonstrate how to calculate necessary sample sizes in order to show the superiority of one classifier over another: this often requires hundreds of statistically independent test samples or is even theoretically impossible. We demonstrate our findings with a data set of ca. 2550 Raman spectra of single cells (five classes: erythrocytes, leukocytes and three tumour cell lines BT-20, MCF-7 and OCI-AML3) as well as by an extensive simulation that allows precise determination of the actual performance of the models in question.

Keywords: Small sample size; Design of experiments; Multivariate; Learning curve; Classification; Training; Validation


Predictive-property-ranked variable reduction in partial least squares modelling with final complexity adapted models: Comparison of properties for ranking by Jan P.M. Andries; Yvan Vander Heyden; Lutgarde M.C. Buydens (pp. 34-45).
Selected variables after variable reduction by the PPRVR-FCAM method, using individual and combined predictor-variable properties.Display Omitted► Variable reduction using the PPRVR-FCAM method is investigated. ► Performance of individual and combined predictor-variable properties is studied. ► Selective and predictive performances of resulting models statistically compared. ► Absolute PLS1 regression coefficient and its significance are most effective.The calibration performance of partial least squares regression for one response (PLS1) can be improved by eliminating uninformative variables. Many variable-reduction methods are based on so-called predictor-variable properties or predictive properties, which are functions of various PLS-model parameters, and which may change during the steps of the variable-reduction process. Recently, a new predictive-property-ranked variable reduction method with final complexity adapted models, denoted as PPRVR-FCAM or simply FCAM, was introduced. It is a backward variable elimination method applied on the predictive-property-ranked variables. The variable number is first reduced, with constant PLS1 model complexity A, until A variables remain, followed by a further decrease in PLS complexity, allowing the final selection of small numbers of variables.In this study for three data sets the utility and effectiveness of six individual and nine combined predictor-variable properties are investigated, when used in the FCAM method. The individual properties include the absolute value of the PLS1 regression coefficient (REG), the significance of the PLS1 regression coefficient (SIG), the norm of the loading weight (NLW) vector, the variable importance in the projection (VIP), the selectivity ratio (SR), and the squared correlation coefficient of a predictor variable with the responsey (COR). The selective and predictive performances of the models resulting from the use of these properties are statistically compared using the one-tailed Wilcoxon signed rank test.The results indicate that the models, resulting from variable reduction with the FCAM method, using individual or combined properties, have similar or better predictive abilities than the full spectrum models. After mean-centring of the data, REG and SIG, provide low numbers of informative variables, with a meaning relevant to the response, and lower than the other individual properties, while the predictive abilities are similar or better. SIG has the best selective ability of all individual and combined properties, while the predictive ability is similar. REG is faster than SIG. This means that variable reduction with the FCAM method is preferably conducted with properties REG or SIG. The selective ability of REG can be improved by combining it with NLW or VIP.

Keywords: Variable reduction; Partial least squares; Predictor-variable properties; Final complexity adapted models


Methodology for the validation of analytical methods involved in uniformity of dosage units tests by E. Rozet; E. Ziemons; R.D. Marini; B. Boulanger; Ph. Hubert (pp. 46-52).
Display Omitted► Methodology to validate methods for uniformity of dosage units tests. ► Valid methods will ensure to make the correct decisions with high probability. ► A Quality by Design compliant validation methodology for UDU assays. ► Analytical Target Profile is defined for UDU assays. ► Application to the validation of an HPLC-UV and NIRS method.Validation of analytical methods is required prior to their routine use. In addition, the current implementation of the Quality by Design (QbD) framework in the pharmaceutical industries aims at improving the quality of the end products starting from its early design stage. However, no regulatory guideline or none of the published methodologies to assess method validation propose decision methodologies that effectively take into account the final purpose of developed analytical methods. In this work a solution is proposed for the specific case of validating analytical methods involved in the assessment of the content uniformity or uniformity of dosage units of a batch of pharmaceutical drug products as proposed in the European or US pharmacopoeias. This methodology uses statistical tolerance intervals as decision tools. Moreover it adequately defines the Analytical Target Profile of analytical methods in order to obtain analytical methods that allow to make correct decisions about Content uniformity or uniformity of dosage units with high probability. The applicability of the proposed methodology is further illustrated using an HPLC-UV assay as well as a near infra-red spectrophotometric method.

Keywords: Quality by Design; Tolerance intervals; Fit for purpose; Content uniformity; Uniformity of dosage units; Analytical Target Profile


Standard addition method applied to the urinary quantification of nicotine in the presence of cotinine and anabasine using surface enhanced Raman spectroscopy and multivariate curve resolution by Mónica B. Mamián-López; Ronei J. Poppi (pp. 53-59).
Display Omitted► Determination of urinary nicotine in the presence of cotinine and anabasine. ► Surface enhanced Raman spectroscopy for analysis of nicotine. ► Multivariate curve resolution in conjunction with standard addition method. ► Determination of nicotine was accomplished with error values less than 10%.In this work, urinary nicotine was determined in the presence of the metabolite cotinine and the alkaloid anabasine using surface enhanced Raman spectroscopy and colloidal gold as substrate. Spectra were decomposed using the multivariate curve resolution-alternating least squares method, and pure contributions were recovered. The standard addition method was applied by spiking urine samples with known amounts of the analyte and relative responses from curve resolution were employed to build the analytical curves. The use of multivariate curve resolution in conjunction with standard addition method showed to be an effective strategy that minimized the need for reagent and time-consuming procedures. The determination of the alkaloid nicotine was successfully accomplished at concentrations 0.10, 0.20 and 0.30μgmL−1 and total error values less than 10% were obtained.

Keywords: SERS; MCR–ALS; Nicotine; Standard addition; Colloidal gold


An absorbing microwave micro-solid-phase extraction device used in non-polar solvent microwave-assisted extraction for the determination of organophosphorus pesticides by Ziming Wang; Xin Zhao; Xu Xu; Lijie Wu; Rui Su; Yajing Zhao; Chengfei Jiang; Hanqi Zhang; Qiang Ma; Chunmei Lu; Deming Dong (pp. 60-68).
Display Omitted► An absorbing microwave μ-SPE device packed with activated carbon was used. ► Absorbing microwave μ-SPE device was made and used to enrich the analytes. ► Absorbing microwave μ-SPE device was made and used to heat samples directly. ► MAE-μ-SPE was applied to the extraction of OPPs with non-polar solvent only.A single-step extraction-cleanup method, including microwave-assisted extraction (MAE) and micro-solid-phase extraction (μ-SPE), was developed for the extraction of ten organophosphorus pesticides in vegetable and fruit samples. Without adding any polar solvent, only one kind of non-polar solvent (hexane) was used as extraction solvent in the whole extraction step. Absorbing microwave μ-SPE device, was prepared by packing activated carbon with microporous polypropylene membrane envelope, and used as not only the sorbent in μ-SPE, but also the microwave absorption medium. Some experimental parameters effecting on extraction efficiency was investigated and optimized. 1.0g of sample, 8mL of hexane and three absorbing microwave μ-SPE devices were added in the microwave extraction vessel, the extraction was carried out under 400W irradiation power at 60°C for 10min. The extracts obtained by MAE-μ-SPE were directly analyzed by GC–MS without any clean-up process. The recoveries were in the range of 93.5–104.6%, and the relative standard deviations were lower than 8.7%.

Keywords: Absorbing microwave micro-solid-phase extraction device; Microwave-assisted extraction; Non-polar solvent; Organophosphorus pesticides


Dumbbell probe-mediated cascade isothermal amplification: A novel strategy for label-free detection of microRNAs and its application to real sample assay by Sai Bi; Yangyang Cui; Li Li (pp. 69-74).
Display Omitted► This assay relies on the circularization of dumbbell probe by target microRNA. ► Rolling circle amplification and autonomous DNA machine are then occurred. ► G-quadruplex is continuously produced to bind hemin to generate color signal. ► High sensitivity, wide dynamic range, and good specificity is achieved.Considering the great significance of microRNAs (miRNAs) in cancer detection and typing, the development of sensitive, specific, quantitative, and low-cost methods for the assay of expression levels of miRNAs is desirable. We describe a highly efficient amplification platform for ultrasensitive analysis of miRNA (taking let-7a miRNA as a model analyte) based on a dumbbell probe-mediated cascade isothermal amplification (DP-CIA) strategy. The method relies on the circularization of dumbbell probe by binding target miRNA, followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking/polymerization/displacement cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal. In terms of the high sensitivity (as low as 1zmol), wide dynamic range (covering 9 orders of magnitude), good specificity (even single-base difference) and easy operation (one probe and three enzymes), the proposed label-free assay is successfully applied to direct detection of let-7a miRNA in real sample (total RNA extracted from human lung tissue), demonstrating an attractive alternative for miRNA analysis for gene expression profiling and molecular diagnostics, particularly for early cancer diagnosis.

Keywords: Analytical methods; Cascade amplification; Label-free detection; MicroRNA


Simple approach to study biomolecule adsorption in polymeric microfluidic channels by Vladimir Gubala; Jonathan Siegrist; Ruairi Monaghan; Brian O’Reilly; Ram Prasad Gandhiraman; Stephen Daniels; David E. Williams; Jens Ducrée (pp. 75-82).
Display Omitted► A simple tool to assess biomolecule adsorption onto the surfaces of microchannels. ► Development for dilution by surface-adsorption based depletion of protein samples. ► It can easily be done using a readily available apparatus like a spin-coater. ► The assessment tool is facile and quantitative. ► Straightforward comparison of different surface chemistries.Herein a simple analytical method is presented for the characterization of biomolecule adsorption on cyclo olefin polymer (COP, trade name: Zeonor®) substrates which are widely used in microfluidic lab-on-a-chip devices. These Zeonor® substrates do not possess native functional groups for specific reactions with biomolecules. Therefore, depending on the application, such substrates must be functionalized by surface chemistry methods to either enhance or suppress biomolecular adsorption. This work demonstrates a microfluidic method for evaluating the adsorption of antibodies and oligonucleotides surfaces. The method uses centrifugal microfluidic flow-through chips and can easily be implemented using common equipment such as a spin coater. The working principle is very simple. The user adds 40L of the solution containing the sample to the starting side of a microfluidic channel, where it is moved through by centrifugal force. Some molecules are adsorbed in the channel. The sample is then collected at the other end in a small reservoir and the biomolecule concentration is measured. As a pilot application, we characterized the adsorption of goat anti-human IgG and a 20-mer DNA on Zeonor®, and on three types of functionalized Zeonor: 3-aminopropyltriethoxysilane (APTES) modified surface with mainly positive charge, negatively charged surface with immobilized bovine serum albumin (BSA), and neutral, hydrogel-like film with polyethylene glycol (PEG) characteristics. This simple analytical approach adds to the fundamental understanding of the interaction forces in real, microfluidic systems. This method provides a straightforward and rapid way to screen surface compositions and chemistry, and relate these to their effects on the sensitivity and resistance to non-specific binding of bioassays using them. In an additional set of experiments, the surface area of the channels in this universal microfluidic chip was increased by precision milling of microscale trenches. This modified surface was then coated with APTES and tested for its potential to serve as a unique protein dilution feature.

Keywords: Antibody adsorption; DNA adsorption; Microfluidics; Polymer substrates; Zeonor; ®


Size exclusion and anion exchange high performance liquid chromatography for characterizing metals bound to marine dissolved organic matter by Natalia García-Otero; Pilar Bermejo-Barrera; Antonio Moreda-Piñeiro (pp. 83-92).
Display Omitted► Fractionation methods for assessing metals bound to marine DOM were developed. ► SEC and AEC with UV detection and hyphenated with inductively coupled plasma-mass spectrometry were used. ► SEC-UV showed marine DOM of molecular weights from 16 to 1kDa. ► Cobalt, manganese, strontium and zinc are bound to marine DOM.Size exclusion chromatography (SEC) followed by anion exchange chromatography (AEC) hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS) was applied for fractionating metals bound to marine dissolved organic matter (DOM). Surface seawater samples (100L) were subjected to tangential flow ultrafiltration (10,000Da cut off) for isolating and pre-concentrating dissolved large molecules. The isolated fraction (retentate) consisted of 1L, which was further freeze-dried and re-dissolved to 250mL with ultrapure water. After HI Trap desalting of the re-dissolved retentate, SEC with UV detection showed marine DOM ranging from 6.5kDa (lower than the permeable volume of the SEC column) to 16kDa. A further characterization of this fraction by AEC with UV detection revealed the existence of four groups of macromolecules exhibiting retention times of 2.3, 2.8, 4.5 and 14.0min. AEC hyphenated with ICP-MS showed the presence of strontium and zinc in the first AE fraction isolated from the SEC fraction; while manganese was found to be bound to the second AE fraction. Cobalt was found to be bound to molecules comprising the third AE fraction.

Keywords: Metal bound to marine dissolved organic matter; Tangential flow ultrafiltration; Size exclusion chromatography; Anion exchange chromatography; Inductively coupled plasma-mass spectrometry


Simultaneous determination of 3-monochloropropane-1,2-diol and acrylamide in food by gas chromatography-triple quadrupole mass spectrometry with coupled column separation by Xiao-min Xu; Hua-li He; Yan Zhu; Liang Feng; Ying Ying; Bai-fen Huang; Hai-tao Shen; Jian-long Han; Yi-ping Ren (pp. 93-99).
MRM chromatograms of 3-MCPD and acrylamide in an instant noodle flavoring sample separated by coupled column.Display Omitted► 3-MCPD and acrylamide were simultaneously detected by GC–MS/MS. ► A coupled column was applied to get sharp and symmetrical peaks. ► MS/MS in MRM was used to suppress matrix interferences and obtain high sensitivity. ► The method was successfully applied to different sample matrices.Both 3-monochloropropane-1,2-diol (3-MCPD) and acrylamide are contaminants found in heat-processed foods and their related products. A quantitative method was developed for the simultaneous determination of both contaminants in food by gas chromatography-triple quadrupole mass spectrometry (GC–MS/MS). The analytes were purified and extracted by the matrix solid-phase dispersion extraction (MSPDE) technique with Extrelut NT. A coupled column (a 3m Innowax combined with a 30m DB-5ms) was developed to separate both compounds efficiently without derivatization. Triple quadrupole mass spectrometry in multiple reaction monitoring mode (MRM) was applied to suppress matrix interference and obtain good sensitivity in the determination of both analytes. The limit of detection (LOD) in the sample matrix was 5μg kg−1 for 3-MCPD or acrylamide. The average recoveries for 3-MCPD and acrylamide in different food matrices were 90.5–107% and 81.9–95.7%, respectively, with the intraday relative standard deviations (RSDs) of 5.6–13.5% and 5.3–13.4%, respectively. The interday RSDs were 6.1–12.6% for 3-MCPD and were 5.0–12.8% for acrylamide. Both contaminants were found in samples of bread, fried chips, fried instant noodles, soy sauce, and instant noodle flavoring. Neither 3-MCPD nor acrylamide was detected in the samples of dairy products (solid or liquid samples) and non-fried instant noodles.

Keywords: 3-Monochloropropane-1,2-diol (3-MCPD); Acrylamide; Heat-processed foods; Flavoring; Gas chromatography-triple quadrupole mass spectrometry (GC–MS/MS); Coupled column
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