Sample Purification Research Articles

HEALTH RISK ASSESSMENT OF POLYCYCLIC AROMATIC HYDROCARBONS

This study investigates the PAH levels in soils around five fuel stations namely: Kwararafa fuel station, Capaduck, Oasis and Ganaco fuel station all in Wukari local government area of Taraba State, Nigeria. The stratified sampling technique was applied in sample collection, extraction and purification by altra-sonication and column chromatography respectively. Quantification of PAHs content by means of GC-MS, The study revealed that the 5.83 μg/kg total composition of the 16 EPA priority polycyclic aromatic hydrocarbons in the test soil was ten times more than the 0.11 μg/kg content of the control soil sample, where dibenz[a,h]anthracene (0.827±0.174 μg/kg) and benz[a]pyrene (0.535±0.174 μg/kg) were the most abundant terms of toxicity based on benzo(a)pyrene-equivalents concentration. Nevertheless there is no human health risk due to inhalation, ingestion and dermal contact respectively.

Rapid determination of 2,4-diaminopyrimidine residues through sample pretreatment using immunomagnetic bead purification along with HPLC–UV

To reduce matrix interference and realize simultaneous detection of multiple homologous compounds (trimethoprim (TMP), diaveridine (DVD), ormetoprim (OMP), baquiloprim (BQP), and aditoprim (ADP) in pig, cattle, chicken, and fish muscles), an immunomagnetic bead (IMB)-based sample purification pretreatment with HPLC–UV was developed. A broad-spectrum monoclonal antibody (mAb, named 14C6) was prepared and conjugated with carboxylic-acid-functionalized magnetic nanoparticles using the active ester method to obtain IMBs for sample purification. The extraction solvent was optimized based on the extraction efficiency. Good linearity was observed for all the five analytes (10–200 μg/kg) with the LOD and LOQ of 5 and 10 μg/kg, respectively. The mean recoveries ranged from 62.5% to 76.9%, while the coefficient of variation was <12.2%. The IMB method afforded greater sample purification and enrichment than those achieved with the SPE column-based conventional method. Hence, the IMB-based sample purification is a useful tool to determine 2,4-diaminopyrimidine residues in edible animal tissues.

Determination of three urinary catecholamines and serotonin by on-line packed-fiber solid-phase extraction

生物单胺包括儿茶酚胺类以及5-羟色胺等,在中枢神经系统中扮演着非常关键的角色,也是临床上诊断神经内分泌肿瘤疾病的重要生物标志物。由于这类单胺类物质的强化学极性导致传统吸附材料对其吸附效果不佳,从复杂生物样本中同时检测更多的生物单胺存在挑战性。该文建立了一种基于聚冠醚纳米纤维在线固相萃取检测尿液中3种儿茶酚胺(多巴胺、肾上腺素、去甲肾上腺素)和5-羟色胺的方法。采用静电纺丝法制备聚二苯并-18-冠-6醚-聚苯乙烯复合纳米纤维(PCE-PS),制成装填纤维的固相萃取(PFSPE)柱,再将PFSPE柱与HPLC进行在线联用。该在线PFSPE-HPLC方法采用双三元泵进行样品富集净化和分析,左泵连接PFSPE柱,进行样品富集净化;右泵连接分析柱进行样品分离检测。控制切换阀的切换,实现样品富集后洗脱至分析柱中分离检测。结果表明,在线PFSPE-HPLC检测尿液儿茶酚胺(多巴胺、肾上腺素、去甲肾上腺素)和5-羟色胺在1~200 ng/mL范围内有良好的线性关系,线性相关系数达0.996以上。3种儿茶酚胺和5-羟色胺的检出限(S/N=3)分别为1和2.5 ng/mL,定量限(S/N=10)分别为2.5和5 ng/mL。空白尿液和实际尿液加标回收率在83.5%~117.7%之间,日内精密度<10%。PCE-PS复合纳米纤维在多次使用后无明显变化,具有良好的稳定性,可重复使用达95次以上。在线PFSPE-HPLC方法能够集样品在线前处理与分析检测于一体,省时省力,实现分析过程的高度自动化。该方法成功应用于尿液中3种儿茶酚胺和5-羟色胺的检测,可以为临床上相关疾病检测诊断和研究提供有力的技术支持。

Radiation synthesis of poly(acrylic acid) nanogels for drug delivery applications – post-synthesis product colloidal stability

Abstract Synthesis of polymer nanogels (NGs) for biomedical applications is considered to be a very promising application in radiation engineering. Under high-dose pulse irradiation of dilute aqueous polymer solution, reactive species generated by water radiolysis can create multiple radicals on each macromolecule and consequently induce intramolecular cross-linking of polymer chains, resulting in NG formation. The obtained products are free from harmful monomers, initiators, and cross-linking agents, which makes them potentially applicable for drug delivery applications. One of the biggest challenges in handling and use of nanoparticles, however, is the colloidal stability, when aqueous suspensions are stored for prolonged periods. Therefore, development of the best protocols for the particular nanocarrier storage is key. To address this need, we have performed the prospective study in which we systematically assessed the influence of various processing and storage scenarios feasible in our lab, on the colloidal stability of the radiation-synthesized poly(acrylic acid) (PAA) NG particles in suspension. This allowed us to choose the optimal way of handling the product after its synthesis. We confirmed that none of the strategies we used and tested are substantially detrimental to our product. Filtration with 0.2-μm filters was proven sufficient for sample purification and prolonged storage in aqueous suspension did not exert a negative effect on the colloidal stability of particles suspension. We have also demonstrated that lyoprotectant-free lyophilization was suitable for our polymer nanoparticles. This is an important fact for further application of particles as nanocarriers for biologically active compounds such as targeting ligands or therapeutic moieties.

Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS.

Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts. Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up. The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) "Guideline on bioanalytical method validation". To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates. Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

Selective precipitation of RNA with linear polyacrylamide

Selective precipitation of RNA is often used in molecular biology as one of the methods for separation of nucleic acids to obtain samples enriched with DNA or RNA molecules alone or for purification of RNA samples. In the present study a simple and fast approach for selective precipitation of RNA with linear polyacrylamide is proposed for the first time. The method is based on the different predispositions of the DNA and RNA molecules to bind with the polyacrylamide. In this process, the linear polyacrylamide is used as the flocculant, collecting RNA particles to form aggregate, which then precipitated at low alcohol concentration. During and after precipitation the temperature is adjusted to maintain high solubility of DNA and other contaminates at given pH, salt and alcohol concentrations on the one hand, and globular state of polyacrylamide, preventing solubility of the RNA-LPA aggregate, on the other hand. The precipitated RNA can be used directly for RT-qPCR assay. The principal advantage of the present approach is the fast and quantitative precipitation of most RNA species from very dilute solutions. This makes it possible to obtain both almost DNA-free RNA and RNA-free DNA samples in one process. Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.2007397 .

Integrated Point-of-Care Molecular Diagnostic Devices for Infectious Diseases.

The global outbreaks of deadly infectious diseases caused by pathogenic microorganisms have threatened public health worldwide and significantly motivated scientists to satisfy an urgent need for a rapid and accurate detection of pathogens. Traditionally, the culture-based technique is considered as the gold standard for pathogen detection, yet it has a long turnaround time due to the overnight culturing and pathogen isolation. Alternatively, nucleic acid amplification tests provide a relatively shorter turnaround time to identify whether pathogens exist in individuals with high sensitivity and high specificity. In most cases, nucleic acid amplification tests undergo three steps: sample preparation, nucleic acid amplification, and signal transduction. Despite the explosive advancement in nucleic acid amplification and signal transduction technologies, the complex and labor-intensive sample preparation steps remain a bottleneck to create a transformative integrated point-of-care (POC) molecular diagnostic device. Researchers have attempted to simplify and integrate the sample preparations for nucleic acid-based molecular diagnostic devices with innovative progress in integration strategies, engineered materials, reagent storages, and fluid actuation. Therefore, understanding the know-how and obtaining truthful knowledge of existing integrated POC molecular diagnostic devices comprising sample preparations, nucleic acid amplification, and signal transduction can generate innovative solutions to achieve personalized precision medicine and improve global health.In this Account, we discuss the challenges of automated sample preparation solutions integrated with nucleic acid amplification and signal transduction for rapid and precise home diagnostics. Blood, nasal swab, saliva, urine, and stool are emphasized as the most commonly used clinical samples for integrated POC molecular diagnostics of infectious diseases. Even though these five types of samples possess relatively correlated biomarkers due to the human body's circulatory system, each shows unique properties and exclusive advantages for molecular diagnostics in specific situations, which are included in this Account. We examine different integrated POC devices for sample preparation, which includes pathogen isolation and enrichment from the crude sample and nucleic acid purification from isolated pathogens. We present the promising on-chip integration approaches for nucleic acid amplification. We also investigate the on-chip integration methods for reagent storage, which is crucial to simplify the manual operation for end-users. Finally, we present several integrated POC molecular diagnostic devices for infectious diseases. The integrated sample preparation and nucleic acid amplification approach reviewed here can potentially impact the next generation of POC molecular home diagnostic chips, which will significantly impact public health, emergency medicine, and global biosecurity.

Apo-H (beta-2-glycoprotein) intact N-glycan analysis by MALDI-TOF-MS using sialic acid derivatization.

Apo-H is a plasma glycoprotein. Nearly 19% of the molecular weight of this protein is composed of glycans. Up- and down-regulation and structural changes in protein glycans provide diagnostic value for disease detection. Here, an efficient, sensitive, and optimized method is developed for Apo-H N-glycans analysis by MALDI-TOF-MS in positive mode. This bioanalytical method includes sample preparation, sample purification, and detection. An Apo-H enrichment method is developed using standard proteins by anti-Apo-H beads followed by enrichment from plasma samples. SDS-PAGE confirms the Apo-H protein enrichment, which is further verified by LC-MS/MS analysis. The lower ionization efficiency of sialylated glycan hampers their analysis by MALDI-MS. For this, stabilization of sialic acids is done by selective derivatization of carboxyl groups to differentiate between α(2,3)- and α(2,6)-linked sialic acids. Glycans are further purified by HILIC-SPE and analyzed by MALDI-MS. Several branched bi- and tri-antennary glycans with fucosylation and sialylation are identified. The reproducibility of the developed method is tested by analyzing multiple replicates of human plasma, where the same glycans are consistently identified. This method could be applied for the Apo-H glycan profiling of large clinical cohorts for diagnostic purposes.

Comprehensive Strategy for Sample Preparation for the Analysis of Food Contaminants and Residues by GC-MS/MS: A Review of Recent Research Trends.

Food safety and quality have been gaining increasing attention in recent years. Gas chromatography coupled to tandem mass spectrometry (GC–MS/MS), a highly sensitive technique, is gradually being preferred to GC–MS in food safety laboratories since it provides a greater degree of separation on contaminants. In the analysis of food contaminants, sample preparation steps are crucial. The extraction of multiple target analytes simultaneously has become a new trend. Thus, multi-residue analytical methods, such as QuEChERs and adsorption extraction, are fast, simple, cheap, effective, robust, and safe. The number of microorganic contaminants has been increasing worldwide in recent years and are considered contaminants of emerging concern. High separation in MS/MS might be, in certain cases, favored to sample preparation selectivity. The ideal sample extraction procedure and purification method should take into account the contaminants of interest. Moreover, these methods should cooperate with high-resolution MS, and other sensitive full scan MSs that can produce a more comprehensive detection of contaminants in foods. In this review, we discuss the most recent trends in preparation methods for highly effective detection and analysis of food contaminants, which can be considered tools in the control of food quality and safety.

Additional Commentary on the Detection and Quantification of Plastic Micro- and Nanoparticles in Tea Samples.

The study of plastic particles, particularly those in the micro-, sub-micro-, and nano-size ranges, within food and beverages has gained increasing interest within recent years. However, many analytical techniques have limits of detection which hinder their use for the study of these particles in these sample matrices. In addition, remaining contaminants from the matrices can interfere with the signals from plastic particles. Thus, great care must be given to sample preparation and data interpretation to ensure accurate results. This study proposes the use of sample purification through chemical digestion protocols to facilitate the study of plastic particles present in tea samples, and serves to highlight technical limitations which must be overcome in future studies.

A method for determination of aldosterone concentrations of six adrenal venous serum samples during a single LC/ESI-MS/MS run using a sextet of Girard reagents

The accurate quantification of aldosterone (ALD) in adrenal tributary venous serum/plasma collected by a super-selective adrenal venous sampling (ssAVS) technique has become a definitive procedure to identify the forms (laterality) of primary aldosteronism (PA) and the location of the affected segment(s), and to subsequently make the treatment decision. In this study, a stable isotope-dilution LC/ESI-MS/MS-based method was developed and validated for the determination of the ALD concentrations of the six ssAVS serum samples during a single run. A sextet of Girard reagents was used to convert ALD to the derivatives having different masses for each sample. Based on the validation tests, satisfactory specificity, precision and accuracy were observed and the matrix effect was found to be acceptable. The developed method was used for the analysis of the ssAVS serum samples from eight PA patients. The batch analysis of the six samples permitted the direct comparison of the ALD concentrations between the samples and enhanced to determine the laterality and location of the affected segment(s). The total analysis time after the sample purification was also reduced to 1/3 for the 24 samples by the sample-sextuplex procedure, which was also an advantage of the developed method.

Solar homogeneous catalysis to the removal of organic matter from slaughterhouse effluents undergone to a prior biological process.

This study shows the effectiveness of the wastewater treatment from a municipal slaughterhouse that has undergone a previous biological treatment applying a sequence of stages, reaching a 75% of elimination of the chemical oxygen demand (COD) using sedimentation in combination with coagulation-flocculation, using 0.5 g/L FeCl3 which is one of the best known inorganic coagulants. Then, the elimination of COD was around 98% adding the Fenton process in which 1,000 mg/L H2O2 and FeSO4 were used. In addition to the COD, other water quality parameters were measured to evaluate the level of purification of the test samples, such as solids of different types, pH, DOC and so on. With the above process, it can be noted that the Fenton process produced a slight improvement in the effluent quality by using a solar concentrator in the now-called photo-Fenton process, reaching around 99% of COD removal (0.36 g/L), 91% of total suspended solids (0.32 g/L) and 89% of dissolved organic carbon (0.20 g/L). These results were the best achieved within a proposed treatment train for this type of complex wastewater. Moreover, this last part of the process adds an improvement by the usage of renewable energy sources such as sunlight.

ECOLOGICAL EFFECTIVE TREATMENT OF INDUSTRIAL WASTEWATER FORMED IN THE OIL-PRODUCING INDUSTRY BY COAGULATION METHOD

Research work has been carried out on deep purification of water samples taken from industrial wastewater formed in the oil-producing industry containing hydrophilic and hydrophobic (oil-in-water and water-in-oil) emulsions from oil and organic suspended substances. Using the coagulation method developed by us, industrial wastewater samples were purified up to 100% from oil and suspended substances under optimal conditions. It has been substantiated that this method is of great economic and environmental importance. The applied coagulant and extractant meet the requirements of environmental safety

Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody-Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry.

The therapeutic efficacy and pharmacokinetics of antibody-drug conjugates (ADCs) in general, and antibody-oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.

Catalytic ferromagnetic gold nanoparticle immunoassay for the detection and differentiation of Mycobacterium tuberculosis and Mycobacterium bovis

A ferromagnetic gold nanoparticle based immune detection assay, exploiting the enhanced signal amplification of inorganic nanozymes, was developed and evaluated for its potential application in the detection of Mycobacterium tuberculosis complex (MTBC) organisms, and simultaneous identification of Mycobacterium bovis. Ferromagnetic gold nanoparticles (Au–Fe3O4 NPs) were prepared and their intrinsic peroxidase-like activity exploited to catalyse 3,3′,5’,5-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). When the Au–Fe3O4 NPs were functionalised by direct coupling with MTBC-selective antibodies, a nanoparticle based immune detection assay (NPIDA) was developed which could detect Mycobacterium tuberculosis (MTB) and differentiate M. bovis. In the assay, the intrinsic magnetic capability of the functionalised Au–Fe3O4 NPs was used in sample preparation to capture target bacterial cells. These were incorporated into a novel immunoassay which used species selective monoclonal antibodies (mAb) to detect bound target. The formation of a blue TMB oxidation product, with a peak absorbance of 370 nm, indicated successful capture and identification of the target. The detection limit of the NPIDA for both MTB and M. bovis was determined to be comparable to conventional ELISA using the same antibodies. Although limited matrix effects were observed in either assay, the NPIDA offers a reduced time to confirmatory identification.This novel NPIDA was capable of simultaneous sample concentration, purification, immunological detection and speciation. To our knowledge, it represents the first immune-based diagnostic test capable of identifying MTBC organisms and simultaneously differentiating M. bovis.

Seaweed Protein Hydrolysates and Bioactive Peptides: Extraction, Purification, and Applications.

Seaweeds are industrially exploited for obtaining pigments, polysaccharides, or phenolic compounds with application in diverse fields. Nevertheless, their rich composition in fiber, minerals, and proteins, has pointed them as a useful source of these components. Seaweed proteins are nutritionally valuable and include several specific enzymes, glycoproteins, cell wall-attached proteins, phycobiliproteins, lectins, or peptides. Extraction of seaweed proteins requires the application of disruptive methods due to the heterogeneous cell wall composition of each macroalgae group. Hence, non-protein molecules like phenolics or polysaccharides may also be co-extracted, affecting the extraction yield. Therefore, depending on the macroalgae and target protein characteristics, the sample pretreatment, extraction and purification techniques must be carefully chosen. Traditional methods like solid–liquid or enzyme-assisted extraction (SLE or EAE) have proven successful. However, alternative techniques as ultrasound- or microwave-assisted extraction (UAE or MAE) can be more efficient. To obtain protein hydrolysates, these proteins are subjected to hydrolyzation reactions, whether with proteases or physical or chemical treatments that disrupt the proteins native folding. These hydrolysates and derived peptides are accounted for bioactive properties, like antioxidant, anti-inflammatory, antimicrobial, or antihypertensive activities, which can be applied to different sectors. In this work, current methods and challenges for protein extraction and purification from seaweeds are addressed, focusing on their potential industrial applications in the food, cosmetic, and pharmaceutical industries.

Accurate and Nonpurified Identification of Extracellular Vesicles Using Dual-Binding Recognition Mode.

Circulating extracellular vesicles (EVs) are promising biomarkers for the early diagnosis and prognosis of cancer in a non-invasive manner. However, the rapid and accurate identification of EVs in complex biological samples is technically challenging, which is attributed to the requirement of extensive sample purification and unsatisfactory detection accuracy due to the disturbance of interfering proteins. Herein, a simultaneous binding of double-positive EV membrane protein-based recognition mode (DRM) is proposed. By the combination of DRM-mediated toehold activation and G-quadruplex DNAZyme-catalyzed etching of Au@Ag nanorods (Au@Ag NRs), we have developed an accurate, non-purified, low-cost, and visual strategy for EV identification. The synchronous binding of double-positive proteins on EV membranes is validated by confocal laser scanning microscopy analysis. This approach exhibits excellent specificity and sensitivity toward EVs ranging from 1.0 × 105 to 1.0 × 109 particles/mL with a detection limit of 6.31 × 104 particles/mL. Moreover, we have successfully realized non-purified EV quantification in complex biological media. In addition, target-initiated catalyzed hairpin assembly (CHA) is integrated with G-quadruplex DNAZyme-catalyzed color variation of Au@Ag NRs; thus, low-background EV detection can be achieved by the naked eye. Furthermore, our strategy is easy to adapt to high-throughput formats by using an automatic microplate reader, which could be expected to meet the requirements for high-throughput detection of clinical samples. With its capacities of rapidness, portability, affordability, high throughput, non-purification, and visual detection, this strategy could provide a practical tool for accurate identification of EVs and early diagnosis of cancer.

Preparative continuous flow electrophoretic instrumentation for purification of biological samples.

We constructed a preparative instrumentation and developed the methods that are based on separation of the samples by bidirectional isotachophoresis/moving boundary electrophoresis in continuous divergent flow. The described instrumentation can be used for a variety of the samples, however, it can be easily optimized and tailored for the specific sample. The trapezoid separation bed from nonwoven textile exhibited minimum adsorption effect for sample and it can be used repeatedly. By the addition of different spacers via separation space inlets, the sections of pH gradient can be modified to enhance the separation. The liquid flow from two inlets positioned on each side of the sample inlet prevented the contact of the sample with anolyte and catholyte at the analysis beginning. One pair of thin electrodes (graphite and stainless-steel) was placed at the separation space output. The electrode products were washed out into drains without disturbing the focusing process. The influence of EOF was managed by tilting the separation bed in the direction from cathodic to anodic side. The components of spirulina supernatant and color pI markers were separated in the pH gradient from 3.9 to 10.1. pH gradient was stable for at least 4.5 h and spirulina supernatant from about 0.12g of dry powder was processed. Compared to other preparative methods used for spirulina separation, the presented method/instrumentation working with a continuous divergent flow had essential advantages. The efficient separation was fast, and no intermediate steps were necessary to obtain liquid fractions with separated components compatible with further biological experiments.

Multiresidue analysis of 15 antibiotics in honey using modified QuEChERS and high performance liquid chromatography-tandem mass spectrometry

Sugars, organic acids, and lipids generally cause strong matrix effects, posing a challenge in the analysis of trace substances in honey. In this study, method involving a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) protocol coupled with high–performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed for the analysis of multiresidue antibiotic in honey samples. Several adsorbents were selected and their abilities to reduce matrix interference and improve detection sensitivity were tested. Zinc oxide (ZnO) proved to be the most effective clean-up adsorbent for the purification of honey samples. Good linearity was found in the concentration range from limits of quantification (LOQs) to 20.0 ng mL–1 for all antibiotics, and for concentration up to 40.0 ng mL–1 for some types of antibiotics, with correlation coefficients exceeding 0.9991. The limits of detection (LODs) ranged from 0.24 to 0.68 μg kg–1, and the LOQs were in the range of 0.80–2.27 μg kg–1. The average recoveries ranged from 81.3 % to 99.0 % at three fortified levels with RSDs below 9.18 %. Twenty-eight honey samples obtained from local markets were analyzed using our optimized method, and lincomycin was detected in one sample.

Removal of Polymerase Chain Reaction Inhibitors by Electromembrane Extraction.

Polymerase chain reaction (PCR) technology has become the cornerstone of DNA analysis. However, special samples (e.g., forensic samples, soil, food, and mineral medicine) may contain powerful PCR inhibitors. High levels of inhibitors can hardly be sufficiently removed by conventional DNA extraction approaches and may result in the complete failure of PCR. In this work, the removal of PCR inhibitors by electromembrane extraction (EME) was investigated for the first time. To demonstrate the universality of the approach, EME formats with and without supported membranes (termed parallel-EME and μ-EME, respectively) were employed, and both anionic [humic acid (HA)] and cationic (Ca2+) PCR inhibitors were used as models. During EME, charged inhibitors in the sample migrate into the liquid membrane in the presence of an electric field and might further leech into the waste solution, while PCR analytes remain in the sample. After EME, the clearance values for HA at 0.2 and 2.5 mg mL-1 were 94 and 85%, respectively, and that for Ca2+ (275 mM) was 63%. Forensic PCR-short tandem repeat (PCR-STR) genotyping showed that EME significantly reduced the interference by HA in PCR-STR analysis and displayed a higher HA purge capability compared to existing methods. Furthermore, by combining EME with liquid-liquid extraction or solid-phase extraction, satisfactory STR profiles were obtained from HA-rich blood samples. In addition, false-negative reports of bacterial detection in mineral medicine and shrimps were avoided after the removal of Ca2+ by μ-EME. Our research demonstrates the great potential of EME for the purification of DNA samples containing high-level PCR inhibitors and opens up a new application direction for EME.