Sample Purification Research Articles

Microplastic sample purification methods - Assessing detrimental effects of purification procedures on specific plastic types

In search of effective, fast, and cheap methods to purify environmental samples for microplastic analysis, scientific literature provides various purification protocols. However, while most of these protocols effectively purify the samples, some may also degrade the targeted polymers. This study was conducted to systematically compare the effects of purification protocols based on acidic, alkaline, oxidative, and enzymatic digestion and extraction via density separation on eight of the most relevant plastic types. It offers insights into how specific purification protocols may compromise microplastic detection by documenting visible and gravimetric effects, analyzing potential surface degradation using Fourier transform infrared spectroscopy (FTIR) and bulk erosion on a molecular level using gel permeation chromatography (GPC). For example, protocols using strong acids and high temperatures are likely to completely dissolve or cause strong degradation to a wide range of polymers (PA, PC, PET, PS, PUR & PVC), while strong alkaline solutions may damage PC and PET. Contrarily, Fenton's reagent, multiple enzymatic digestion steps, as well as treatment with a zinc chloride solution frequently used for density-separation, do not degrade the eight polymers tested here. Therefore, their implementation in microplastic sample processing may be considered an essential stepping-stone towards a standardized protocol for future microplastics analyses.

Use of sorbents to improve water quality in the production of reconstituted dairy products

Ensuring the safety of dairy products is one of the components of integrated food safety. The safety of dairy products is directly related to the organization of production control carried out when assessing the quality of water of the centralized drinking water supply system. The article discusses the feasibility of using natural and modified zeolites to ensure water safety. The physical and chemical properties of natural zeolites of the Taizhuzgen and Shankanai deposits of Kazakhstan, the possibility of their use to improve the quality of water in the production of reconstituted dairy products were investigated. The efficiency of water sample purification with modified zeolite of the Shankanai deposit for heavy metal ions was: Pb2+ – 79%, Cd2+ - 98%, Fe2+ - 77%, depending on the initial impurity concentration. It was found that the studied sorbents can be used to improve the quality of water in the production of reconstituted dairy products

Determination of retinol and carotenoids in selected Malaysian food products using high-performance liquid chromatography (HPLC)

The presented method is focused on simultaneous determination of retinol and two carotenoids (lutein, β-carotene) in selected Malaysian food products. A Dionex Ultimate 3000 Series High-Performance Liquid Chromatography system equipped with two Dionex Acclaim Polar Advantage II C18 columns was utilized in this study. Simultaneous elution of retinol, lutein and β-carotene was achieved within 45 min with gradient profile of methanol and ultra-pure water (95:5–100:0–95:5) programmed at 0.8 mL/min flow rate and 30 °C. Retinol was acquired at 325 nm while lutein and β-carotene were acquired at 440 nm. Limits of detection and quantification of retinol and lutein were at 0.10, 0.34 µg/mL, and 0.08, 0.26 µg/mL; respectively while higher levels were observed for β-carotene at 1.06 and 3.54 µg/mL. Linearity test indicated R2 of 0.996–0.999, along with high accuracy (71.92–116.29%) and excellent repeatability for all analytes. Extraction efficiency was determined using standard addition into representative food product with recovery obtained in the range of 61.86–116.90% for all three analytes. The developed in-house HPLC method incorporates an enclosed solid-phase extraction system that is believed to further improve sample purification and analytes detection. On the whole, the developed on-line SPE-HPLC technique and efficient sample pre-treatment is deemed a reliable approach for simultaneous determination of retinol, lutein and β-carotene, as analyzed among the selected Malaysian food products. Additionally, the described procedure could also benefit routine analysis of these analytes in other food matrices or for determination of other analytes with similar chemistry and faster elution.

UPLC-MS/MS assay for the simultaneous determination of catecholamines and their metabolites at low pg/mg in rat/mouse striatum

Catecholamines and their metabolites act as neurotransmitters in the brain and are important for nervous system function. In the current work, a highly selective and sensitive UPLC-MS/MS assay was developed for quantitation of six catecholamines and their metabolites, including dopamine, norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindolacetic acid from rat and mouse striatum as pharmacodynamic biomarkers to support neuroscience and pharmaceutical research. A fit-for-purpose strategy for method development, assay qualification and study support were adopted for this assay. A surrogate matrix (brain homogenizing solution absent of targeted analytes) was used for preparation of calibration samples and certain levels of quality control samples to avoid interference from endogenous baselines. Homogenized rodent striatum was derivatized by dansyl chloride to enhance the sensitivity, followed by liquid-liquid extraction with ethyl acetate in 96-well plate format. The lower limit of quantitation (LLOQ) was 0.2 ng/mL in tissue homogenate, equivalent to 3.2 pg/mg in brain tissue, which could be further reduced to ten times lower by changing the re-dissolving and injecting volume in the last sample purification step. Acceptable accuracy, precision, linearity, specificity, recovery, and matrix effect was obtained. Bench-top stability (2 h), freeze-thaw stability (3 cycles at −20 °C), and − 80 °C storage stability (up to 51 days) in both tissue homogenate and surrogate matrix along with autosampler stability (60 h at 4°C) all met acceptance criteria. This assay was successfully applied to measure the six analytes in striatum from mice treated with the neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an animal model of Parkinsonism for which dosing protocols can vary widely, and further confirmed the metabolic pathway of neurotoxicity by the quantification of catecholamine metabolites. Our study is the first detailed the step-by-step recovery and pointed out the key factors for the assay to simultaneously quantify these six neurotransmitters in rodent striatum with superior sensitivity.

Qualitative and quantitative determination of tilmicosin in poultry eggs by gas chromatography tandem mass spectrometry after derivatization with acetic anhydride

A novel GC-MS/MS analytical method was established for the qualitative and quantitative determination of tilmicosin in poultry (Jinghai yellow chicken, Gaoyou duck and Yangzhou goose) eggs. The method was based on LLE and SPE for sample extraction and purification. Pyridine and acetic anhydride were used for the derivatization reaction. When tilmicosin was added to blank poultry egg samples at the LOQ and 75 μg/kg, 150 μg/kg, and 300 μg/kg, the recoveries ranged from 72.80% to 88.75%, the intraday and interday RSDs ranged from 2.31% to 4.56% and 3.29%-5.61%, respectively, and the LODs and LOQs ranged from 3.8 to 5.6 μg/kg and 8.4-10.5 μg/kg, respectively. These results confirmed that the parameters of this novel method meet the requirements of the FAO & WHO (2014) for veterinary drug residue testing. Poultry egg samples purchased from the local market were analysed according to the established method and only one egg sample was found to contain 18.9 μg/kg of tilmicosin.

Monitoring of Aflatoxin M1 in Various Origins Greek Milk Samples Using Liquid Chromatography Tandem Mass Spectrometry

Aflatoxin M1(AFM1), a major metabolite of Aflatoxin B1(AFB1), has been identified as a potential contaminant in dairy products. Because of its possible carcinogenicity, the legislation limits as set by Commission Regulation (EC) No. 1881/2006 are very strict, namely 0.050 μg kg−1 in milk and 0.025 μg kg−1 in infant formulas. To meet these requirements, a sensitive and accurate method was developed, employing liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Ιmmunoaffinity columns (R-Biopharm) were used for sample purification and preconcentration of the analyte of interest. The quantification of AFM1 was conducted using fortified milk samples, while Aflatoxin B2 (AFB2) was used as an internal standard (IS). The method was validated in terms of linearity, precision, trueness, limits of detection and quantification and uncertainty. The performance criteria for the method were evaluated based on European Commission Regulation (EC) No. 401/2006 and its most recent amendment, as well as the suggested criteria for revision by the EU Reference Laboratory for Mycotoxins and Plant Toxins. The recovery was in the range of 77.9–81.0% for all fortification levels (0.025–0.050–0.075 μg kg−1), with RSDR values (Relative Standard Deviation of intermediate precision) ranging from 6.1% to 12%. The method’s detection and quantification limits were 0.0027 μg kg−1 and 0.0089 μg kg−1, respectively. The occurrence of AFM1 was investigated in 40 samples of different animal origin (cow, goat and sheep milk) provided by Greek producers.

Simultaneous determination of fourteen β2-agonist enantiomers in food animal muscles by liquid chromatography coupled with tandem mass spectrometry

Illegal drug residues in animal derived foods are closely related to human's life and health. Studies on illegal drug residues and the metabolism, such as β2-agonists in animals have attracted more and more attention. In most cases, β2-agonists are suppliedand used astheracemate. The metabolic process and distribution of the two enantiomers in animal tissues are different. Therefore, it is very necessary to develop a simple and fast method for chiral resolution of these drugs in animal tissues. In this paper, a reliable resolution and determination method was presented using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for fourteen enantiomers of seven β2-agonist racemates, clenbuterol (CLE), salbutamol (SAL), cimaterol (CIM), terbutaline (TER), clorprenaline (CLO), tulobuterol (TUL), penbuterol (PEN) in pork, beef, and lamb muscle samples. The samples were added the internal standard solution (IS) and extracted in the alkaline medium with acetonitrile. The further sample purification was accomplished through MCX solid phase extraction cartridge. Chromatographic chiral separation was carried out on a VancoShell chiral column (100 mm × 4.6 mm, 2.7 μm) with an isocratic mobile phase consisting of methanol and 10 mmol mL−1 ammonium formate aqueous solution (85:15, v/v). Under the optimized conditions, the resolution (R) of CIM was 2.0, CLE and PEN were 1.5, the others were all greater than 1.0. Enantiomeric determination was performed in the positive electrospray ionization mode using multiple reaction monitoring (MRM). The correlation coefficient (r) in the range of 0.2–25.0 μg L−1 was above 0.993. The average recoveries at the three spiking levels ranged from 95.3% to 117.7% with the relative standard deviation (RSD) lower than 15%. The limit of detection (LOD) and the limit of quantification (LOQ) of β2-agonist enantiomers was 0.2 μg kg−1 and 0.5 μg kg−1 respectively. The method was successfully applied in the analysis and evaluation of β2-agonist enantiomers in positive food animal muscle samples, CLE, SAL, TEB and CIM enantiomers were detected. The concentrations of the corresponding enantiomers were in the range of 1.06–17.3 μg kg−1, the lowest enantiomer fraction (EF) value was 0.42, and the highest value was 0.69. The work is expected to provide a method for chiral separation and enantiomeric determination of the further study of pharmacology, toxicity and residue elimination of β2-agonist enantiomers.

Separation of halloysite/kaolinite mixtures in water controlled by sucrose addition: The influence of the attractive forces on the sedimentation behavior

In this work, we propose an easy strategy for the separation of halloysite/kaolinite mixtures in sucrose aqueous solution. Preliminarily, we investigated the influence of the sucrose addition on the colloidal stability of kaolinite nanoplates and halloysite nanotubes (HNTs) dispersed in water. Dynamic Light Scattering (DLS) measurements revealed that the HNTs aqueous mobility is dependent on the sucrose concentration, while the ζ-potential is negligibly affected by the addition of the carbohydrate in the aqueous solvent. On the other hand, any variations on the surface charge and dynamic behavior of kaolinite were detected in the presence of sucrose. The obtained ζ-potential and DLS results were useful to interpret the sedimentation kinetics of kaolinite and halloysite dispersions. In particular, the peculiar sedimentation behavior of halloysite were discussed by considering the screening effect of sucrose on the attractive interactions between the nanotubes. The latter was evaluated by the calculation of the corresponding Hamaker constants. On this basis, we estimated that the optimization of the separation between kaolinite and halloysite might be achieved in 10 wt% sucrose aqueous solution. This hypothesis was confirmed by the separation experiments, which exploited the different sedimentation behavior of halloysite and kaolinite. Thermogravimetric experiments on the separated material and kaolinite/halloysite composites with variable composition allowed us to determine the efficiency of the separation protocol. To this purpose, the differential thermogravimetric curves were accurately analysed by using split Gaussian functions. In conclusion, this paper represents a further step in the purification of natural halloysite samples exploiting the ability of sucrose to control the attractive forces of the clay nanotubes as well as the colloidal stability of kaolinite nanoplates in aqueous solvent.

Pinpointing Alkane Chain Length, Saturation, and Double Bond Regio- and Stereoisomers by Liquid Interfacial Plasmonic Enhanced Raman Spectroscopy.

The lipids with a rich diversity of isomers face a formidable challenge in comprehensive structural analysis. The commonly used mass spectrometry-based techniques usually require a considerable number of molecules with sophisticated chemical derivatization or ion mobility separation, but the co-existing of structurally similar isomers often makes the distinction impossible. Here, we develop an alternative powerful liquid/liquid interfacial surface-enhanced Raman spectroscopy (SERS) strategy at normal temperature and pressure without any sources of ionization or radiation. This strategy generates high-resolution fingerprints in molecular chain length, C═C position, saturation, and regio- and stereoisomers of both glycerides and fatty acids and requires only trace amounts of molecules down to 1 ppb to achieve discrimination and exhibits great potentials to push the identification capability to trace levels or even the single-molecule level. According to experimental data and theoretical simulations, these targets have the amphiphilic and emulsifying properties, exhibit ordered molecular orientation and adsorption patterns, promote the co-assembly with plasmonic nanoarrays at the immiscible liquid/liquid interface, and consequently amplify the detection sensitivity. As a contrast, the typical SERS based on solid/air interfacial plasmonic nanoarrays faces the intrinsic bottleneck of extremely weak intensity and indistinguishable spectral fingerprints of lipid molecules. The vibrational fingerprints exhibit a rich range of well-resolved absorption features that are clearly diagnostic for fine structural changes and pave a new way for straightforward measurement without laborsome sample purification, enrichment, or complex derivatization. Although challenging, its unprecedented resolving power expands the potentials of SERS, serving as an ultimate analytical method to provide insights into the detailed structural features of other lipids under facile conditions in the future.

Microfluidic Applications in Drug Development: Fabrication of Drug Carriers and Drug Toxicity Screening.

Microfluidic technology has been highly useful in nanovolume sample preparation, separation, synthesis, purification, detection and assay, which are advantageous in drug development. This review highlights the recent developments and trends in microfluidic applications in two areas of drug development. First, we focus on how microfluidics has been developed as a facile tool for the fabrication of drug carriers including microparticles and nanoparticles. Second, we discuss how microfluidic chips could be used as an independent platform or integrated with other technologies in drug toxicity screening. Challenges and future perspectives of microfluidic applications in drug development have also been provided considering the present technological limitations.

A pump-free microfluidic device for fast magnetic labeling of ischemic stroke biomarkers.

This research proposes a low-cost and simple operation microfluidic chip to enhance the magnetic labeling efficiency of two ischemic stroke biomarkers: cellular fibronectin (c-Fn) and matrix metallopeptidase 9 (MMP9). This fully portable and pump-free microfluidic chip is operated based on capillary attractions without any external power source and battery. It uses an integrated cellulose sponge to absorb the samples. At the same time, a magnetic field is aligned to hold the target labeled by the magnetic nanoparticles (MNPs) in the pre-concentrated chamber. By using this approach, the specific targets are labeled from the beginning of the sampling process without preliminary sample purification. The proposed study enhanced the labeling efficiency from 1h to 15min. The dynamic interactions occur in the serpentine channel, while the crescent formation of MNPs in the pre-concentrated chamber, acting as a magnetic filter, improves the biomarker-MNP interaction. The labeling optimization by the proposed device influences the dynamic range by optimizing the MNP ratio to fit the linear range across the clinical cutoff value. The limits of detection (LODs) of 2.8ng/mL and 54.6ng/mL of c-Fn measurement were achieved for undiluted and four times dilutions of MNP, respectively. While for MMP9, the LODs were 11.5ng/mL for undiluted functionalized MNP and 132ng/mL for four times dilutions of functionalized MNP. The results highlight the potential use of this device for clinical sample preparation and specific magnetic target labeling. When combined with a detection system, it could also be used as an integrated component of a point-of-care platform.

Electrophoretic µPAD for Purification and Analysis of DNA Samples.

In this work, the fabrication and characterization of a simple, inexpensive, and effective microfluidic paper analytic device (µPAD) for monitoring DNA samples is reported. The glass microfiber-based chip has been fabricated by a new wax-based transfer-printing technique and an electrode printing process. It is capable of moving DNA effectively in a time-dependent fashion. The nucleic acid sample is not damaged by this process and is accumulated in front of the anode, but not directly on the electrode. Thus, further DNA processing is feasible. The system allows the DNA to be purified by separating it from other components in sample mixtures such as proteins. Furthermore, it is demonstrated that DNA can be moved through several layers of the glass fiber material. This proof of concept will provide the basis for the development of rapid test systems, e.g., for the detection of pathogens in water samples.

Middle-up quantification of therapeutic monoclonal antibodies in human plasma with two dimensional liquid chromatography high resolution mass spectrometry: Adalimumab as a proof of principle

Next generation human therapeutic monoclonal antibodies (t-mAbs) are harder to quantify with the widely used bottom-up tryptic digestion method. Due to their homology with endogenous immunoglobulins, there is a lack of unique and stable ‘signature’ peptides that can be targeted. Middle-up two dimensional liquid chromatography high resolution mass spectrometry (2D-LC-HRMS), targeting the entire light chain, was examined as an alternative. Adalimumab (ADM) was successfully quantified in human plasma after Melon® Gel sample purification, followed by orthogonal separation on a weak cation exchange (WCX) and reversed phase column. Charge and hydrophobicity were used to separate ADM from the polyclonal immunoglobulin background. HRMS with its high resolution and exact mass was able to isotopically resolve the ADM light chain and to provide another separation dimension on the basis of mass to charge ratio. Using the targeted single ion monitoring (T-SIM) with multiplex (MSX) option, three ADM light chain precursors, 2341.80, 2129.00, and 1951.68 m/z, and one internal standard precursor 2146.39 m/z, were measured simultaneously. The Melon® Gel sample purification was found to be very efficient in removing plasma proteins that would otherwise interfere with chromatographic separation and ionization. The linearity of the method for the analysis of ADM was excellent (R2=0.999) between 1 – 128 mg/L with an LLOQ signal to noise ratio (S/N) of 10. Within-run and between-run precision and accuracy were in concordance with the EMA guideline. Cross-validation of the 2D-LC-HRM method with the standard peptide LC-MS/MS method showed a good agreement (R2 = 0.86) between the methods. However, there was a bias present, possibly due to charge variant ADM formation over time. Since the presented 2D-LC-HRMS method is able to measure only the native form of ADM, it is able to provide a measure of the active form of ADM in patients.

Zwitterionic Polymer Electroplating Facilitates the Preparation of Electrode Surfaces for Biosensing.

Surface chemistry critically affects the diagnostic performance of biosensors. An ideal sensor surface should be resistant to nonspecific protein adsorption, yet be conducive to analytical responses. Here a new polymeric material, zwitterionic polypyrrole (ZiPPy), is reported to produce optimal surface condition for biosensing electrodes. ZiPPy combines two unique advantages: the zwitterionic function that efficiently hydrates electrode surface, hindering nonspecific binding of hydrophobic proteins; and the pyrrole backbone, which enables rapid (<7min), controlled deposition of ZiPPy through electropolymerization. ZiPPy-coated electrodes show lower electrochemical impedance and less nonspecific protein adsorption (low fouling), outperforming bare and polypyrrole-coated electrodes. Moreover, affinity ligands for target biomarkers can be immobilized together with ZiPPy in a single-step electropolymerization. ZiPPy-coated electrodes are developed with specificity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The prepared sensor detects SARS-CoV-2 antibodies in human saliva down to 50ng mL-1 , without the need for sample purification or secondary labeling.

Simultaneous Quantification of Chloramphenicol, Thiamphenicol, Florfenicol, and Florfenicol Amine in Animal and Aquaculture Products Using Liquid Chromatography-Tandem Mass Spectrometry.

The accumulation of antimicrobial residues in edible animal products and aquaculture products could pose health concerns to unsuspecting consumers. Hence, this study aimed to develop a validated method for simultaneous quantification of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in beef, pork, chicken, shrimp, eel, and flatfish using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Primary-secondary amine (PSA) and MgSO4 were used for sample purification. The analytes were separated on a reversed-phase analytical column. The coefficients of determination for the linear matrix-matched calibration curves were ≥0.9941. Recovery rates ranged between 64.26 and 116.51% for the four analytes with relative standard deviations (RSDs) ≤ 18.05%. The calculated limits of detection (LODs) and limits of quantification (LOQs) were 0.005–3.1 and 0.02–10.4 μg/kg, respectively. The developed method was successfully applied for monitoring samples obtained from local markets in Seoul, Republic of Korea. The target residues were not detected in any tested matrix. The designed method was versatile, sensitive, and proved suitable for quantifying residues in animal-derived products.

Backdoor Defence for Voice Print Recognition Model Based on Speech Enhancement and Weight Pruning

Voice print recognition is one of the most mature biometric authentication technologies, and the application of deep neural networks (DNNs) has led to a significant improvement in the accuracy of voice print recognition. However, DNN models can be attacked by backdoor attackers, which poses a serious threat to the security of model for voice print recognition. In this paper, a method is proposed for backdoor defence of voice print recognition model based on speech enhancement and weight pruning. Firstly, input samples are perturbed by superimposing various speech patterns, and the backdoor samples are determined based on the randomness (entropy value) of the prediction classes with perturbed inputs from a given deployment model (malicious or benign). Secondly, the backdoor samples are fed into a network (Deep Complex Convolution Recurrent Network) dedicated for speech enhancement, with which the backdoor samples can be denoised by removing the backdoor noise. Finally, the model is pruned using an automatic progressive weight pruning algorithm, which can avoid the accuracy degradation caused by neurons pruning. Experimental results on the AISHELL speech dataset show that the method not only reduces the success rate of backdoor attacks, but also greatly realizes the purification of the backdoor samples.

Methods to Analyze the Redox Reactivity of Plant Proteins.

Proteins can be covalently modified by a broad range of highly reactive chemicals and redox mechanisms. Reversible redox-mediated post-translational modifications of sensitive cysteine thiol groups in proteins impact protein characteristics such as interaction behavior and activity state. Evaluating the response of proteins to redox perturbation or reactive chemical species is critical for understanding the underlying mechanisms involved and their contribution to plant stress physiology. Here we provide a detailed workflow that includes procedures for (i) purification, processing, and analysis of protein samples with redox agents, (ii) determining redox-modulated monomer to oligomer transitions using size exclusion chromatography, and (iii) activity assays for monitoring the impact of redox agents on purified enzymes and in crude extracts from plants subjected to oxidative stress. We exemplified how to apply several of the methods discussed for analyzing redox-sensing metallopeptidases, such as thimet oligopeptidases. We anticipate that these protocols should find broad applications in monitoring biochemical properties of other classes of redox-sensitive plant proteins.

Ce and Nd stable isotope purification and determination of geological samples by MC-ICP-MS

A TODGA resin column was used to isolate Ba, La, Ce, Pr and Nd from geological samples.

Analytical methods for pesticide residues determination in propolis and propolis-based products

Abstract As a result of massive bee deaths in recent years, beekeeping is raising concerns about the presence of pesticides in propolis which is considered as a safe product. The paper is focused on the analysis of bee propolis and propolis-based products using various chromatographic techniques using mass spectrometry detection predominantly. An important part of the work is an overview concerning methods of sample preparation, extraction, and purification of extracts, followed by separation and detection techniques. Positive findings of contaminants and their concentrations in propolis samples and propolis products were evaluated. Sorption based techniques such as matrix solid phase dispersion and solvent-based extraction techniques are frequently applied for propolis analysis in connection with chromatographic techniques. Liquid-based extractions, such as the QuEChERS extraction technique (Quick, Easy, Cheap, Effective, Rugged, Safe), combine extraction by solvent with several ways of extract cleaning using combinations of salts and sorbents, primary secondary amine, MgSO4, NaCl, graphitized carbon, EMR-lipid, florisil, or octadecylsilane-modified silica gel. Other extraction techniques were reviewed. The most significant problem to be considered in pesticides detection is the topic of matrix effects, which have to be solved for each sample analysis with special care.

Generating Sequencing-Based DNA Methylation Maps from Low DNA Input Samples.

Reduced representation bisulfite sequencing (RRBS) is a technique used for assessing genome-wide DNA methylation patterns in eukaryotes. RRBS was introduced to focus on CpG-rich regions that are likely to be of most interest for epigenetic regulation, such as gene promoters and enhancer sequence elements (Meissner et al., Nature 454:766-770, 2008). This "reduced representation" lowers the cost of sequencing and also gives increased depth of coverage, facilitating the resolution of more subtle changes in methylation levels. Here, we describe a modified RRBS sequencing (RRBS-seq) library preparation. Our protocol is optimized for generating single base-resolution libraries when low input DNA is a concern (10-100ng). Our protocol includes steps to optimize library preparation, such as using deparaffinization solution (when formalin-fixed material is used), and a replacement of gel size-selection with sample purification beads. The described protocol can be accomplished in 3days and has been successfully applied to tissues or cells from different organisms, including formalin-fixed tissues, to yield robust and reproducible results.