Rapid electrophoretic recovery of DNA from dried blood spots

Large‐scale genetic screening of neonatal dried blood spots for episomal DNA has a great potential to lower patient mortality and morbidity through early diagnosis of primary immunodeficiencies. However, DNA extraction from the surface of dried blood spots remains one of the most time consuming, costly, and labor‐intensive parts of DNA analysis. In the present study, we developed and optimized a rapid methodology using only 50 V and heat to extract episomal DNA from dried blood spots prepared from diagnostic cord blood samples. This electric field DNA extraction is the first methodology to use an electric field to extract episomal DNA from a dried blood spot. This 25‐minute procedure has one of the lowest times for the extraction of episomal DNA found within the literature and this novel procedure not only negates the need for costly treatment and wash steps, but reduces the time of manual procedures by more than 30 min while retaining the 75–80% of the yield. Combined with real‐time PCR, this novel method of electric field extraction not only provides an effective tool for the large scale genetic analysis of neonates, but a key step forward in the simplification and standardization of diagnostic testing.     

Mixed‐mode SPE for a multi‐residue analysis of benzodiazepines in whole blood using rapid GC with negative‐ion chemical ionization MS

A sensitive and selective method for simultaneous quantitation of 15 benzodiazepines in human whole blood using rapid GC with negative‐ion chemical ionization MS is proposed. A mixed‐mode cation‐exchange polymeric sorbent was used for SPE. Different extraction solvents or mixtures of solvents of different compositions for elution of the adsorbed analytes, and washing steps for eliminating interferences in the column were tested. Analytes were eluted from the column using 5% v/v NH₄OH in methanol. A derivatization step using different silylation reagents, time, and temperature was tested. Extracts from SPE were silylated by a mixture of N‐(tert‐butyldimethylsilyl)‐N‐methyltrifluoroacetamide, acetonitrile, ethyl acetate, and subjected to gas chromatographic analysis. The LODs of 15 benzodiazepines in whole blood samples ranged from 0.24–0.62 ng mL^?1. The RSDs of samples used for three different quality control concentration levels were lower than 7.0%, and the accuracy ranged from 89.5 to 110.5%. The results show that the developed method is accurate, sensitive, selective, and very fast. Finally, the applicability of this method for determination of trace concentrations of several benzodiazepines in real blood samples has been demonstrated.     

Determination of amphetamine and methadone in human urine by microextraction by packed sorbent coupled directly to mass spectrometry: An alternative for rapid clinical and forensic analysis†

Speed and low cost, together with regulatory approval, are the most important requirements of clinical assays. Therefore, a fast and automated on‐line sample preparation method is essential for the routine analysis of biological samples. Microextraction by packed sorbent is an option for optimal sample preparation due to its easy automation, minimal requirements for the sample and elution solvent volumes, elimination of evaporation and reconstitution steps, and ability to integrate sample preparation and injection into one step. The use of effective sample preparation steps circumvents the need for chromatographic separation and therefore allows more rapid and less expensive sample analysis in clinical and forensic practice. Two biologically active compounds, amphetamine and methadone, were chosen as representative drugs of abuse for the application of microextraction by packed sorbent coupled directly to mass spectrometry. The developed method was validated, with the results confirming the suitability of the combination of these techniques for the analysis of biological samples. The approach was confirmed to be appropriate for use in clinical and forensic practice with regard to cost and time requirements for analysis.     

人牙齿中锶的特效树脂分离及其同位素测定

采用锶特效树脂(Sr-Spec)建立了快速分离富集人牙齿中微量元素锶,并测定87Sr/86Sr的有效方法。采用HNO3-HClO4体系消解牙齿样品,以8mol/L HNO3为介质上柱,8mol/L HNO3淋洗,0.05mol/LHNO3洗脱,收集淋洗液,蒸干;采用正热电离质谱法进行87Sr/86Sr的测定。结果表明,利用Sr-Spec树脂,不仅能将锶与基质中大量钙分离,并能有效分离同位素测定中干扰元素铷。本方法可以缩短分离时间,提高分离效率,减少试剂用量,降低实验空白。采用本方法对陕南地区人牙齿牙釉质中锶进行分离,测得的87Sr/86Sr值在0.710948～0.711037之间。     

Mitochondrial DNA control region typing from highly degraded skeletal remains by single-multiplex next-generation sequencing

Poor nuclear DNA preservation from highly degraded skeletal remains is the most limiting factor for the genetic identification of individuals. Mitochondrial DNA (mtDNA) typing, and especially of the control region (CR), using next-generation sequencing (NGS), enables retrieval of valuable genetic information in forensic contexts where highly degraded human skeletal remains are the only source of genetic material. Currently, NGS commercial kits can type all mtDNA-CR in fewer steps than the conventional Sanger technique. The PowerSeq CRM Nested System kit (Promega Corporation) employs a nested multiplex-polymerase chain reaction (PCR) strategy to amplify and index all mtDNA-CR in a single reaction. Our study analyzes the success of mtDNA-CR typing of highly degraded human skeletons using the PowerSeq CRM Nested System kit. We used samples from 41 individuals from different time periods to test three protocols (M1, M2, and M3) based on modifications of PCR conditions. To analyze the detected variants, two bioinformatic procedures were compared: an in-house pipeline and the GeneMarker HTS software. The results showed that many samples were not analyzed when the standard protocol (M1) was used. In contrast, the M3 protocol, which includes 35 PCR cycles and longer denaturation and extension steps, successfully recovered the mtDNA-CR from highly degraded skeletal samples. Mixed base profiles and the percentage of damaged reads were both indicators of possible contamination and can provide better results if used together. Furthermore, our freely available in-house pipeline can provide variants concordant with the forensic software.     

Qualitative screening for volatile organic compounds in human blood using solid‐phase microextraction and gas chromatography‐mass spectrometry

A fast and simple screening procedure using solid‐phase microextraction and gas chromatography‐mass spectrometry (SPME‐GC‐MS) in full‐scan mode for the determination of volatile organic compounds (VOC) is presented. The development of a fast and simple screening technique for the simultaneous determination of various volatiles is of great importance, because of their widespread use, frequent occurrence in forensic toxicological questions and the fact that there is often no hint on involved substances at the crime scene. To simulate a screening procedure, eight VOC with different chemical characteristics were chosen (isoflurane, halothane, hexane, chloroform, benzene, isooctane, toluene and xylene). To achieve maximum sensitivity, variables that influence the SPME process, such as type of fiber, extraction and desorption temperature and time, agitation and additives were optimized by preliminary studies and by means of a central composite design. The limits of detection and recoveries ranged from 2.9 µg/l (xylene) to 37.1 µg/l (isoflurane) and 7.9% (chloroform) to 61.5% (benzene), respectively. This procedure can be used to answer various forensic and toxicological questions. The short time taken for the whole analytical procedure may make its eventual adoption for routine analysis attractive. Copyright © 2010 John Wiley & Sons, Ltd.     

MALDI‐TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varieties

A procedure for identification of malting barley varieties using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of ethanol‐soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI‐TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry‐based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd.     

Mixed hemimicelle magnetic dispersive solid‐phase extraction using carbon‐coated magnetic nanoparticles for the determination of tramadol in urine samples

Ionic liquid carbon‐coated magnetic nanoparticles were successfully applied as an adsorbent in a mixed hemimicelle magnetic dispersive solid‐phase extraction method for the determination of tramadol from urine samples coupled with high‐performance liquid chromatography with UV‐vis detection. The significant parameters affect the extraction efficiency including type and amount of adsorbent, sample volume, pH, ionic strength, type and amount of elution solvent, time of extraction and desorption, time of ionic liquid loading on the adsorbent and stirring rate were studied and optimized. The proposed method provided a fast, straightforward, environmentally friendly and adsorbent recyclable approach for tramadol analysis. The linear range for the tramadol determination was from 100 to 1500 ng/mL. Precisions and accuracies were within 6%. The applicability of the proposed method in clinical trial was tried successfully on determination of tramadol in addicted subjects under tramadol therapy. The mean percent recovery of the patient samples was 94%. The results proved that the proposed method could be applied in clinical and forensic laboratories for determination of tramadol from biological urine samples.     

Development of an analytical procedure for quantifying the underivatized neurotoxin β-N-methylamino-l-alanine in brain tissues

The cyanotoxin β-methylamino-l-alanine (BMAA) has received renewed attention as an environmental risk factor for sporadic cases of amyotrophic lateral sclerosis (ALS) (Nunn et al., Brain Res 410:375–379, 1987). The aim of the present study was to develop and to validate an analytical procedure that allows the quantification of native BMAA and of its natural isomer, 2,4 diaminobutyric acid (DAB), in brain tissues. An analytical procedure was previously reported by our group for the determination of underivatized BMAA in environmental samples. It included a step of sample clean-up by solid phase extraction (SPE) with a mixed-mode sorbent and the analyses were performed by LC/MS-MS using hydrophilic interaction chromatography and multiple reactions monitoring scan mode. As brain tissues have a higher lipid content, the crucial step of sample clean-up had been optimized by evaluating the efficiency of the addition of a liquid/liquid extraction step prior to the SPE procedure or alternatively, of washing steps to the SPE extraction procedure. The efficiency was checked by visualizing the complexity of the resulting chromatograms in LC/MS and their performance by using spiked brain samples. The optimized analytical procedure, including a washing step with cyclohexane to the SPE with a recovery yield close to 100 %, was validated using the total error approach and allowed the quantification of BMAA in a concentration level ranging from 20 to 1,500 ng/g in brain samples. Finally, the feasibility of implementation of this procedure was verified in human brain samples from two patients who died of ALS.     

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.     

Paper‐Origami‐Based Multiplexed Malaria Diagnostics from Whole Blood

We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.     

Integrated massively parallel sequencing of 15 autosomal STRs and Amelogenin using a simplified library preparation approach

Massively parallel sequencing (MPS) technologies, also termed as next‐generation sequencing (NGS), are becoming increasingly popular in study of short tandem repeats (STR). However, current library preparation methods are usually based on ligation or two‐round PCR that requires more steps, making it time‐consuming (about 2 days), laborious and expensive. In this study, a 16‐plex STR typing system was designed with fusion primer strategy based on the Ion Torrent S5 XL platform which could effectively resolve the above challenges for forensic DNA database‐type samples (bloodstains, saliva stains, etc.). The efficiency of this system was tested in 253 Han Chinese participants. The libraries were prepared without DNA isolation and adapter ligation, and the whole process only required approximately 5 h. The proportion of thoroughly genotyped samples in which all the 16 loci were successfully genotyped was 86% (220/256). Of the samples, 99.7% showed 100% concordance between NGS‐based STR typing and capillary electrophoresis (CE)‐based STR typing. The inconsistency might have been caused by off‐ladder alleles and mutations in primer binding sites. Overall, this panel enabled the large‐scale genotyping of the DNA samples with controlled quality and quantity because it is a simple, operation‐friendly process flow that saves labor, time and costs.     

Determination of trihalomethanes in soil matrices by simplified quick,easy, cheap,effective, rugged and safe extraction and fast gas chromatography with electron capture detection

A method based on QuEChERS extraction is proposed for the determination of trihalomethanes (chloroform, bromodichloromethane, dibromocloromethane and bromoform) in soil samples. The new version of QuEChERS adapted to soil samples consists of liquid extraction with ethyl acetate, the addition of water to moisten the samples, salting-out partitioning of the water with anhydrous MgSO₄, and direct injection of the organic extract, obtained after the centrifugation step, into the gas chromatograph. This simplified extraction procedure maintains the advantages of the original method and avoids some steps, making the final procedure simpler, faster, and cheaper, with the consequent reduction in errors associated with sample manipulation. The experimental conditions of the analytical method, based on fast gas chromatography (FGC) and micro-electron capture detection (μECD), were optimized. The column and oven program used allowed fast separation of the compounds in less than 4 min and the total analysis cycle time was as short as 10 min. The existence of a matrix effect was checked and the analytical conditions of the method were studied in a fortified garden soil sample. The highly sensitive and selective detector used afforded to detection limits in the order of ng/kg for the target compounds. To validate the proposed method two certified reference materials (CRMs) were analyzed.     

Toward a microchip-based solid-phase extraction method for isolation of nucleic acids

A silica-based solid-phase extraction system suitable for incorporation into a microchip platform (nu-total analytical system; nu-TAS) would find utility in a variety of genetic analysis protocols, including DNA sequencing. The extraction procedure utilized is based on adsorption of the DNA onto bare silica. The procedure involves three steps: (i) DNA adsorption in the presence of a chaotropic salt, (ii) removal of contaminants with an alcohol/water solution, and (iii) elution of the adsorbed DNA in a small volume of buffer suitable for polymerase chain reaction (PCR) amplification. Multiple approaches for incorporation of this protocol into a microchip were examined with regard to extraction efficiency, reproducibility, stability, and the potential to provide PCR-amplifiable DNA. These included packing microchannels with silica beads only, generating a continuous silica network via sol-gel chemistry, and combinations of these. The optimal approach was found to involve immobilizing silica beads packed into the channel using a sol-gel network. This method allowed for successful extraction and elution of nanogram quantities of DNA in less than 25 min, with the DNA obtained in the elution buffer fraction. Evaluation of the eluted DNA indicated that it was of suitable quality for subsequent amplification by PCR.     

Fast and efficient extraction methods for the analysis of polychlorinated biphenyls and polybrominated diphenyl ethers in biological matrices

This paper describes fast and simple extraction methods for the determination of polychlorinated biphenyls and polybrominated diphenyl ethers in biological matrices. Four extraction protocols were tested. The first protocol used microwave-assisted extraction combined with two purification steps. The second one was similar, except that microwave-assisted extraction was replaced by accelerated solvent extraction. The third one combined extraction/purification by accelerated solvent extraction with final purification on a silica gel column. The last one combined microwave-assisted extraction with purification on an acidic silica gel column. The protocols were tested on various matrices: a spiked matrix, two certified matrices (SRM 2977, WMF 01), and natural matrices (mysids and fish). All of the protocols produced good performance in terms of recovery and reproducibility. The two last protocols showed promising results in terms of applicability to natural matrices, as they required a minimum of sample handling and minimal amounts of solvent and time. These methods allowed at least 24 samples to be handled per day, and could easily be used for routine analysis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fast analysis of glycosides based on HKUST‐1‐coated monolith solid‐phase microextraction and direct analysis in real‐time mass spectrometry

Glycosides are a kind of highly important natural aromatic precursors in tobacco leaves. In this study, a novel HKUST‐1‐coated monolith dip‐it sampler was designed for the fast and sensitive analysis of trace glycosides using direct analysis in real‐time mass spectrometry. This device was prepared in two steps: in situ polymerization of monolith in a glass capillary of dip‐it and layer‐by‐layer growth of HKUST‐1 on the surface of monolith. Sufficient extraction was realized by immersing the tip to solution and in situ desorption was carried out by plasma direct analysis in real time. Compared with traditional solid‐phase microextraction protocols, sample desorption was not needed anymore, and only extraction conditions were needed to be optimized in this method, including the gas temperature of direct analysis in real time, extraction time, and CH₃COONH₄ additive concentration. This method enabled the simultaneous detection of six kinds of glycosides with the limits of detection of 0.02–0.05 μg/mL and the linear ranges covering two orders of magnitude with the limits of quantitation of 0.05–0.1 μg/mL. Moreover, the developed method was applied for the glycosides analysis of three tobacco samples, which only took about 2 s for every sample.     

Simultaneous determination of β2‐agonists in human urine by fast‐gas chromatography/mass spectrometry: method validation and clinical application

A fast screening protocol was developed and validated for the simultaneous determination of 15 β₂‐agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid–liquid extraction, followed by derivatization of the extract and detection of β₂‐agonists trimethylsilyl‐derivatives by fast‐gas chromatography/electron impact–mass spectrometry (fast‐GC/EI‐MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra‐ and inter‐assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level. Fast‐GC/MS sequences, based on the use of short columns, high carrier‐gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/h. High MS‐sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments. Copyright © 2009 John Wiley & Sons, Ltd.     

Microextraction sample preparation techniques in forensic analytical toxicology

Sample preparation is a critical step in forensic analytical toxicology. Different extraction techniques are employed with the goals of removing interferences from the biological samples, such as blood, tissues and hair, reducing matrix effects and concentrating the target analytes, among others. With the objective of developing faster and more ecological procedures, microextraction techniques have been expanding their applications in the recent years. This article reviews various microextraction methods, which include solid‐based microextraction, such as solid‐phase microextraction, microextraction by packed sorbent and stir‐bar sorptive extraction, and liquid‐based microextraction, such as single drop/hollow fiber‐based liquid‐phase microextraction and dispersive liquid–liquid microextraction, as well as their applications to forensic toxicology analysis. The development trend in future microextraction sample preparation is discussed.     

Evaluation of the protective capabilities of nucleosome STRs obtained by large‐scale sequencing

Partial DNA profiles are often obtained from degraded forensic samples and are hard to analyze and interpret. With in‐depth studies on degraded DNA, an increasing number of forensic scientists have focused on the intrinsic structural properties of DNA. In theory, nucleosomes offer protection to the bound DNA by limiting access to enzymes. In our study, we performed large‐scale DNA sequencing on nucleosome core DNA of human leucocytes. Five nucleosome short tandem repeats (STRs) were selected (including three forensic common STRs (i.e. TPOX, TH01, and D10S1248) and two unpublished STRs (i.e. AC012568.7 and AC007160.3)). We performed a population genetic investigation and forensic genetic statistical analysis of these two unpublished loci on 108 healthy unrelated individuals of the HeBei Han population in China. We estimated the protective capabilities of five selected nucleosome loci and MiniFiler? loci with artificial degraded DNA and case samples. We also analyzed differences between sequencing results and software predicted results. Our findings showed that nucleosome STRs were more likely to be detected than MiniFiler? loci. They were well protected from degradation by nucleosomes and could be candidates for further nucleosome multiplex construction, which would increase the chances of obtaining a better balanced profile with fewer allelic drop‐outs.     

Resolving human DNA mixtures with F‐108 polymer and capillary electrophoresis single‐strand conformational polymorphism analysis

Current technologies have increased the sensitivity for analyzing forensic DNA samples, especially those considered “touch samples.” Because of this, there has been an increase in the number of forensic mixtures–two or more contributors within a single sample–submitted to the crime laboratories. Therefore, the need to resolve these mixtures has increased as well. Several technologies are currently utilized, but many of them are time consuming and do not resolve the entire profile. Therefore, CE‐Single‐Strand Conformational Polymorphisms coupled with the Pluronic F‐108 polymer was assessed for its ability to resolve human forensic mixtures. This technique has been able to detect sequence variation, such as single nucleotide polymorphism in short tandem repeat loci, such as D7S820 and vWA. Samples were first analyzed with the Performance Optimized Polymer‐7, and mixtures created from samples that shared alleles. These samples were sequenced to detect single base‐pair mutations and evaluated with the F‐108 and CE‐Single Strand Conformational Polymorphism analysis. Results from this study indicated the method would serve as a valuable screening tool to detect base sequence variation between individuals when they share alleles in a mixture and before using Massive Parallel Sequencing technology to distinguish which bases differ.