Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples

Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories. It offers higher resolution to address most problems of human identification, greater efficiency and potential ability to interrogate very challenging forensic casework samples. In this study, a trial set of DNA samples was artificially degraded by progressive aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample and control sample 2800 M, to test the performance and reliability of the ForenSeqTM DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and 0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the NGS approach even from sub-optimal amounts of high quality DNA. The degraded samples showed a very limited number of reads/sample, from 2.9\u201310.2 folds lower than the ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was impossible to assign up to 78.2% of the genotypes in the degraded samples as the software identified the corresponding loci as \u201clow coverage\u201d ( 50x). Amplification artifacts such as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded samples. However, the ForenSeqTM DNA Sequencing kit, on the Illumina MiSeq, was able to generate data which led to the correct typing of 5.1\u201344.8% and 10.9\u201358.7% of 58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed higher chances to be typed correctly compared to the STRs. This NGS approach showed very promising results in terms of ability to recover genetic information from heavily degraded DNA samples for which the conventional PCR/CE approach gave no results. The frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of the degraded samples. However, these results suggest that further validation studies and a definition of interpretation criteria for NGS data are needed before implementation of this technique in forensic genetics

liquid phase microextraction techniques combined with chromatography analysis a review

Sample pretreatment is the first and the most important step of an analytical procedure. In routine analysis, liquid–liquid microextraction (LLE) is the most widely used sample pre-treatment technique, whose goal is to isolate the target analytes, provide enrichment, with cleanup to lower the chemical noise, and enhance the signal. The use of extensive volumes of hazardous organic solvents and production of large amounts of waste make LLE procedures unsuitable for modern, highly automated laboratories, expensive, and environmentally unfriendly. In the past two decades, liquid-phase microextraction (LPME) was introduced to overcome these drawbacks. Thanks to the need of only a few microliters of extraction solvent, LPME techniques have been widely adopted by the scientific community. The aim of this review is to report on the state-of-the-art LPME techniques used in gas and liquid chromatography. Attention was paid to the classification of the LPME operating modes, to the historical contextualization of LPME applications, and to the advantages of microextraction in methods respecting the value of green analytical chemistry. Technical aspects such as description of methodology selected in method development for routine use, specific variants of LPME developed for complex matrices, derivatization, and enrichment techniques are also discussed

Solid phase microextraction techniques used for gas chromatography: A review

In the last decade, the development and adoption of greener and sustainable microextraction techniques have been proved to be an effective alternative to classical sample preparation procedures. In this review, 10 commercially available solid-phase microextraction systems are presented, with special attention to the appraisal of their analytical, bioanalytical, and environmental engineering. This review provides an overview of the challenges and achievements in the application of fully automated miniaturized sample preparation methods in analytical laboratories. Both theoretical and practical aspects of these environment-friendly preparation approaches are discussed. The application of chemometrics in method development is also discussed. We are convinced that green analytical chemistry will be really useful in the years ahead. The application of cheap, fast, automated, "clever", and environmentally safe procedures to environmental, clinical, and food analysis will improve significantly the quality of the analytical data

Determination of glycine and threonine in topical dermatological preparations

In the present study, a single HPLCmethod was developed for the determination of glycine and threonine in cicatrizants. Two different preparations of a cream and an ointment, and the corresponding bandages, onto which the formulations were applied, were studied. The method involved matrix solubilisation with dichloromethane, liquid–liquid isolation of gly and thr with aqueous 1N NaOH, and derivatization with phenylisothiocyanate. Reversed-phase HPLC separation was carried out by gradient elution with 20mM aqueous NaClO4 and acetonitrile (from 90% to 30% aqueous NaClO4 in 10 min) on a LiChrospher® 100 RP-18 cartridge (125mm×4.6 mm). Analytes were determined with a UV detector set at 245 nm. Quantitation was accomplished by internal standardization with methionine. Linearity was studied in the range 60–120% of the concentrations expected for gly and thr (viz. for gly from 200 to 400gml−1, and for thr from 100 to 200gml−1). In reference aqueous samples, linear correlation (r) was better than 0.99 for gly and thr, while in spiked matrix samples r ranged from 0.97 to 0.98. Recoveries were in the 95–105% interval, and precision (CV%, N= 6)was better than 5% for both analytes either in cream, ointment or bandages. The method was successfully used for the quality control of topical dermatological preparations

Determination of Quinic acid in beverages and fungi by Hydrophylic Interaction Liquid Chromatography (HILIC) and UV detection at low wavelength

The present study reports on the development and application of a hydrophilic interaction liquid chromatography (HILIC) method for the separation of quinic acid (QA) in food matrixes. QA is a cyclohexanecarboxylic acid (mw 192 Da) evidenced in Quina (cinchona bark), cranberry, coffee beans and coffee beans-derived products [1,2]. QA is naturally found free or bound in chlorogenic acids and it is thought to be involved in the perceived acidity of coffee and fruit-derived beverages. QA determination therefore can be of interest in the quality control and authenticity testing of foods and beverages derived from fruits, beans and berries. Although QA levels should be expected to be quite commonly measured in vegetable and fruit extracts, this is rarely done. The reason for this lack of data on QA could be that this molecule is difficult to analyze in HPLC because of its high polarity, hydrophilicity and lack of chromophores [3-6]. In this study a simple HILIC-UV method was developed for the determination of QA using a ZIC-cHILIC 150x2.1mm, 3µm p.s. column at room temperature, with gradient elution using a pH 6.0 phosphate buffer and UV detection at 200nm. Validation of the method was carried out on cranberry juice. QA levels were measured in cranberry juice and in several other fruit juices, vegetable beverages, and fung

Hydrophilic interaction chromatography in food matrices analysis: an updated review

This review focuses on the most recent papers (from 2011 to submission date in 2017) dealing with the analysis of different organic components in foods (i.e. nucleobases, nucleosides, nucleotides, uric acid, and creatinine, amino acids and related compounds, choline-related compounds and phospholipids, carbohydrates, artificial sweeteners and polyphenolic compounds), using hydrophilic interaction liquid chromatography (HILIC) combined with different detection techniques. For each compound class, the investigated food matrices are grouped per: foods of animal origin, vegetables, fruits and related products, baby food, and other matrices such as drinks and mushrooms/fungi. Furthermore, the main advantages of HILIC chromatography respect to the other commonly used techniques are discussed

Determination of the Sugar Content in Commercial Plant Milks by Near Infrared Spectroscopy and Luff-Schoorl Total Glucose Titration

Thirty-nine samples of plant milks (rice, soy, oat, barley, spelt, quinoa, almond, and a variety of wheat called kamut) were analyzed for their reducing sugars content by NIR spectroscopy, using the Luff-Schoorl official method as reference to build the calibration models. The amount of reducing sugars, expressed as grams of glucose/100 mL of beverage, ranged from 0.5 g/100 mL (soy) to 7.6 g/100 mL (rice). Both partial least squares (PLS) and interval-partial least squares regression (iPLS) were used to build multivariate calibration models, testing different spectra preprocessing methods. The performance in prediction of the best calibration model was evaluated on an external test set of nine randomly selected samples (RMSEP = 0.98 g/100 mL, R2 PRED = 0.84), and its statistical significance was assessed using a randomization t test based on Monte Carlo simulations. The results obtained suggest that NIR spectroscopy can be a valid alternative to the laborious reference titrimetric method for the determination of total glucose content in plant milks