Precipitation Protocol Research Articles

Lung Transplant Recipients with SARS-CoV-2 Infection Induce Circulating Exosomes with SARS-CoV-2 Spike Protein S2 Which Are Immunogenic in Mice

PurposeExosomes are nanosized vesicles released by cells into body fluids. We have demonstrated the presence of circulating exosomes containing viral antigens in lung transplant recipients (LTxR) undergoing rejection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an important risk factor for LTxR undergoing immunosuppression. Our goal is to determine whether exosomes with SARS-CoV-2 spike protein are induced in LTxR with SARS-CoV-2 infection and exosomes are immunogenic in mice, inducing immune responses to the spike proteinMethodsWe analyzed 67 patients with SARS-CoV-2 infection for the induction of circulating exosomes with SARS-CoV-2 spike protein. Exosomes were isolated from plasma by an exosome precipitation protocol followed by 0.2 micron filtration and size determination by NanoSight300. Exosomes were first analyzed by western blot with specific antibodies to SARS-CoV-2 spike and its nucleoprotein. Eluted proteins from the gel were analyzed by mass spectrometry. Exosomes were subjected to transmission electron microscopy (TEM) to detect spike and nucleocapsid antigens. To determine the immunogenicity of isolated exosomes, C57BL/6 mice were immunized with exosomes carrying SARS-CoV-2 spike protein.ResultsExosomes from SARS-CoV-2 infected LTxR expressed SARS-CoV-2 spike protein S2 and increased levels of RNA related to SARS-CoV-2. Peptides specific for SARS-CoV-2 spike protein in exosomes were confirmed by mass spectroscopy. TEM also revealed the expression of spike protein and nucleocapsid antigens on the exosome surface. Mice immunized with exosomes carrying the spike protein, developed antibodies to SARS-CoV-2 spike antigens. Severe inflammation and lesions were also demonstrated in the lungs of mice immunized with exosomes carrying SARS-CoV-2 spike protein. Splenic lymphocytes from mice immunized with exosomes carrying SARS-CoV-2 spike antigen also demonstrated increased frequency of T-cells which are spike protein antigen specific and secreting IFN-γ and TNF-α .ConclusionSARS-CoV-2 infected LTxR induce circulating exosomes with spike protein and nucleic acids related to SARS-CoV-2. Since the induced exosomes are highly immunogenic, we propose that the exosomes induced by SARS-CoV-2 will have immunological consequences relevant to the COVID19 disease process.

Recovery and Purification of Fumaric Acid from Fermented Oil Palm Empty Fruit Bunches Using a Simple Two-Stage Precipitation Procedure

Oil palm empty fruit bunches (EFBs) are an attractive lignocellulosic material that can be used as a cheap renewable feedstock to produce organic acids and many other value-added products. This research is aimed at investigating the potential of steam-exploded oil palm EFBs for the production of fumaric acid, a food additive widely used for flavor and preservation, through a separate hydrolysis and fermentation process using the selected fungal isolate Rhizopus oryzae K20. To develop an efficient method for the recovery and purification of fumaric acid from fermented oil palm EFBs, a two-stage precipitation protocol was employed, followed by an activated carbon-mediated polishing step to remove contaminants. After these two processes were accomplished, a recovery yield of 81.2% and a purity of 83.5% were achieved.

In-situ H2O2 production for tetracycline degradation on Ag/s-(Co3O4/NiFe2O4) visible light magnetically recyclable photocatalyst

Few investigations have reported appreciable photocatalytic H2O2 formation from pure water with the in situ degradation of an organic pollutant. We have prepared a magnetically recyclable Ag/s-(Co3O4/NiFe2O4) type II heterojunction photocatalyst that produced considerable H2O2 from pure water under visible light irradiation. Furthermore, the H2O2 produced by this process could efficiently degrade tetracycline. A multi-step precipitation protocol was followed for the preparation of this photocatalyst. Components for photocatalyst construction were chosen using band positions and oxygen adsorption properties. Large-scale molecular dynamics simulations were used to investigate the affinity of a component surface toward oxygen adsorption in an aqueous medium. XPS analysis suggested that Ag nanostructures on the Co3O4/NiFe2O4 composite as electron-rich reaction centers for oxygen reduction·H2O2 production on this photocatalyst was tested under various control conditions. The presence of an electron donor further enhanced both H2O2 production and tetracycline degradation rates.

Global mapping of RNA G-quadruplexes (G4-RNAs) using G4RP-seq.

Guanine-rich RNAs can fold into four-stranded structures, termed G-quadruplexes (G4-RNAs), and participate in a wide range of biological processes. Here we describe in detail a G4-RNA-specific precipitation (G4RP) protocol, which enables the transcriptomic profiling of G4-RNAs. The G4RP protocol consists of a chemical cross-linking step, followed by affinity capture with a G4-specific probe, BioTASQ. G4RP can be coupled with sequencing to capture a comprehensive global snapshot of folded G4-RNAs. This method can also be used to profile induced changes (i.e., through G4 ligand treatments) within the G4-RNA transcriptome. The entire protocol can be completed in 1-2 weeks and can be scaled up or down depending on the specific experimental goals. In addition to the protocol details, we also provide here a guide for optimization in different laboratory setups.

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry.

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

Salt-Mediated Organic Solvent Precipitation for Enhanced Recovery of Peptides Generated by Pepsin Digestion.

Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.

Validation and Clinical Application of a New Liquid Chromatography Coupled to Mass Spectrometry (HPLC-MS) Method for Dalbavancin Quantification in Human Plasma

Dalbavancin (DBV) is an intravenous long-acting second-generation glycolipopeptide antibiotic with high efficacy and excellent tolerability, approved for use in the treatment of Gram-positive skin and skin structure infections (ABSSSI). Nevertheless, little is known about its pharmacokinetic/pharmacodynamic (PK/PD) properties in real life, which is also due to technical challenges in its quantification in human plasma, preventing an effective application of therapeutic drug monitoring (TDM). In fact, DBV has a high affinity to plasma proteins, possibly resulting in poor recovery after extraction procedure. The aim of this study was to validate a simple, cheap and reliable HPLC-MS method for use in TDM, in accordance with FDA and EMA guidelines. The optimized protein precipitation protocol required 50 μL of plasma, while chromatographic analysis could be performed in 12 min/sample. This method fulfilled the guidelines requirements and then, it was applied for routine DBV TDM in patients receiving off-label high doses (two 1500 + 1500 mg weekly infusions instead of 1000 + 500 mg), with normal renal function or undergoing hemodialysis: continuous hemodiafiltration caused a relevant reduction in DBV exposure, while intermittent dialysis showed comparable DBV concentrations with those of patients with normal renal function. This confirmed the eligibility of the presented method for use in TDM and its usefulness in clinical practice.

Synthesis of Al doped CoP2/rGO composite and its high electrocatalytic activity for hydrogen evolution reaction

A series of Al-doped CoP2/rGO composites have been fabricated by in-situ precipitation and phosphorization protocol. The well-ordered arrangement and widely adjustable mole ratio of cations within Co–Al layered double hydroxide (CoAl-LDH) make it suitable as a precursor for phosphiding and generating evenly dispersed Al element within CoP2 structure. The effect of Al doping content on the catalytic activities for hydrogen evolution reaction thus could be discussed in detail. Electrocatalytic performance indicates that the doping of Al element within CoP2 structure could efficiently prevent the oxidation of phosphide on the surface, and thus resulting in the high electrocatalytic activity for hydrogen evolution reaction (HER). Among all the Al-doped CoP2/rGO materials, the composite with the Co/Al mole ratio of 2/1 presents the highest HER activity and stability whatever under acidic or basic reaction conditions, which could be comparable to the most reported HER electrocatalysts and even the commercial 20% Pt/C. At the current density of −10 ​mA ​cm−2 in 0.5 ​M ​H2SO4, the overpotential is only 59 ​mV with a Tafel slope of 73.2 ​mV dec−1. Interestingly, the Al content could be dissolved under basic media and resulting in a porous structured electrode. Only the overpotential of 60 ​mV is needed to get the current density of −10 ​mA ​cm−2 in 1 ​M KOH.

A rapid and simple protocol for concentration of SARS-CoV-2 from sewage

The aim of this study was to set up a simple protocol to concentrate SARS-CoV-2 from sewage, which can be implemented in laboratories with minimal equipment resources. The method avoids the need for extensive purification steps and reduces the concentration of potential inhibitors of RT-qPCR contained in sewage. The concentration method consists of a single step, in which a small volume (40 mL) of sewage sample is incubated with polyaluminum chloride (PAC)(0.00045 N Al3+ final concentration). Virus particles adsorbed to the precipitate are collected by low-speed centrifugation, after which the recovered pellet is resuspended with a saline buffer. PAC-concentrated samples are stable for at least one week at 4 °C. Therefore, they may be sent refrigerated to a diagnosis center for RNA extraction and RT-qPCR for SARS-CoV-2 RNA detection if the lab does not have such capabilities. The PAC concentration method produced an average shift of 4.5-units in quantification cycle (Cq) values compared to non-concentrated samples, indicating a 25-fold increase in detection sensitivity. The lower detection limit corresponded approximately to 100 viral copies per ml. Kappa index indicated substantial agreement between PAC and polyethylene glycol (PEG) precipitation protocols (k = 0.688, CI 0.457−0.919). This low-cost concentration protocol could be useful to aid in the monitoring of community circulation of SARS-CoV-2, especially in low- and middle-income countries, which do not have massive access to support from specialized labs for sewage surveillance.

Standardization of a protein extraction protocol for the proteomic analysis of the leaves of Oryza sativa L. cv. BRS AG

AbstractThe rice cultivar BRS AG (Oryza sativa L.) is a cereal developed to serve as a raw material for ethanol production or animal feed, contributing to an increasing energy demand and the economic sustainability of the rice sector. However, only few studies have reported this new cultivar. As proteomic analysis is crucial in understanding the physiological and adaptive behavior of BRS AG rice leaves under biotic and abiotic stresses, the present study compared the following three precipitation protocols for the protein extraction of leaf tissue in two‐dimensional electrophoresis analysis: phenol method, trichloroacetic acid‐acetone method, and a third method developed in our laboratory (BIOPRO). The efficiency and repeatability of the protein extraction protocols were evaluated by two‐dimensional electrophoresis analysis. Our results indicate that the BIOPRO protocol was the most efficient in protein extraction as compared to the efficiency of other protocols. We detected a greater amount of protein (15.44 μg/μL) and 414 spots, and the BIOPRO protocol provided the best results regarding reproducibility, focusing, resolved spots, the intensity of spots, and single spot detection. This novel protocol is useful for extracting high‐quality proteins from leaves and presents an efficient removal of contaminants, which is directly linked to a better resolution for two‐dimensional electrophoresis analysis.

Improved thermal stability and luminescence properties of SrSi2O2N2:Eu2+ green phosphor by a heterogeneous precipitation protocol for solid-state lighting applications

The SrSi2O2N2:Eu2+ green-emitting phosphor, as a member of oxynitride phosphors, could greatly enhance the color quality of the white-light conversed by single yellow-emitting phosphor. However, SrSi2O2N2:Eu2+ phosphors prepared by traditional solid-state ways normally consist hard agglomerates with unevenly dispersed particles and therefore suppress the luminescence properties. This research proposes a heterogeneous precipitation protocol where precipitation process is introduced, in order to produce a precursor in the first phase. And in the second phase, a high-temperature solid-state process is adopted for obtaining the final product. The main results show that, for an optimized SrSi2O2N2:Eu2+ phosphor prepared using our protocol, (1) the SrSi2O2N2:Eu2+ particles are faceted and with smooth faces and (2) the phosphor photoluminescence, external quantum efficiency, stability against thermal exposure, and physical stability consistently outperform the current commercially used product. This research essentially provides an economic way for production of solid-state lighting with enhanced color quality.

Purification of polysaccharides from Phellinus linteus by using an aqueous two-phase system and evaluation of the physicochemical and antioxidant properties of polysaccharides in vitro

In this study, Phellinus linteus polysaccharides (PLPS) and proteins were simultaneously separated from P. linteus mycelia by using an aqueous two-phase system (ATPS) based on choline chloride ([Chol]Cl)/K2HPO4, and the physicochemical and antioxidant properties of PLPS after ATPS extraction were evaluated. Results demonstrated that the maximal extraction efficiencies of 68.53% ± 0.29% PLPS and 82.37% ± 0.41% proteins were obtained when the cholinium-based ATPS contained 68.9% K2HPO4, 20% [Chol]Cl, 10.0 mg mL−1 crude water extract (1.0 mL), and distilled water (4.0 mL) at shaking time and temperature of 30 min and 21.2 °C, respectively. Compared with C-PLPS obtained using traditional ethanol precipitation and isolation protocols, PLPS had higher carbohydrate content (63.58% ± 1.12%), lower molecular weight (15.2 kDa, 80%), different monosaccharide compositions, and showed similar preliminary structural characterizations. Moreover, PLPS exhibited more evident scavenging effects on free radicals and in vitro antioxidant activities than C-PLPS. Therefore, the method of [Chol]Cl/K2HPO4 ATPS can be developed as an effective strategy for the separation/purification of highly bioactive polysaccharides.

AgI/CuWO4 Z-scheme photocatalyst for the degradation of organic pollutants: Experimental and molecular dynamics studies

While the knowledge of the adsorption properties of components of a composite heterogeneous photocatalyst is critical to its applicability to a particular reaction, there has been little research in this direction. The present research is on the development of AgI/CuWO4 nanocomposites that photocatalytically degraded ciprofloxacin and rhodamine B in an aqueous medium under visible light irradiation. The nanocomposites were prepared by a step-wise precipitation protocol. XPS analysis and active species trapping experiments demonstrated that the photocatalysis proceeded by a Z-scheme mechanism. Large scale aqueous medium molecular dynamics simulations showed that oxygen and CIP adsorb on the AgI part, while water interacts intensely with the CuWO4 component. Information from experimental and molecular dynamics studies was combined to arrive at the photocatalysis mechanism.

Efficient Molecular Biological Manipulations with Improved Strategies Based on Novel Escherichia coli Vectors.

In this study, some novel plasmids have been constructed for flexible and zero-background molecular cloning, more efficient expression, and purification of proteins with improved strategies. The plasmids pANY4-pL18-ccdB and pANY4-pR18/pL18-ccdB have different promoters in the complementary DNA strands. Therefore, recombinant plasmids for either isopropyl-β-d-thiogalactoside-induced or temperature-induced protein expression could be simultaneously constructed in a single molecular cloning process for parallel comparison. Intriguingly, the mutated pL18 and pR18/pL18 promoters performed similar to or even better than the T7 promoter when used for promoting the expression of the GFP or pfLamA enzyme. Moreover, the plasmid pANY8 containing the His-elastin-like polypeptide (ELP)-intein multifunctional tag was constructed, and special purification protocol was designed to obtain purified proteins without the requirement of time-consuming dialysis steps to remove imidazole and high concentration of salt ions. Additionally, the urea-based denaturation and refolding processes can be conveniently integrated into the ELP-mediated precipitation protocol for purification of insoluble inclusion bodies, omitting the time-consuming dialysis steps.

Determination of seventeen free amino acids in human urine and plasma samples using quadruple isotope dilution mass spectrometry combined with hydrophilic interaction liquid chromatography – Tandem mass spectrometry

Taking into account the growing demand for new analytical procedures that are appropriate for analysis of complex biological samples with increased sensitivity, accuracy and precision, a novel analytical method was described for the determination of underivatized amino acids in human plasma and urine samples. The presented analytical procedure involved the direct analysis of urine samples and the analysis of plasma samples followed by a simple protein precipitation protocol. Samples were analyzed using a simple and fast chromatographic method developed for the determination of 17 different amino acids by liquid chromatography - tandem mass spectrometry. The limit of detection and quantification values for amino acids were ranged between 0.03–2.26 µmol kg−1 and 0.09–7.54 µmol kg−1. Matrix effects of plasma and urine on the quantification of analytes were determined by spiking experiments. The accuracy of method was evaluated by matrix matching and quadruple isotope dilution strategies. Excellent accuracy and precision were obtained with the use isotope labeled amino acids demonstrating the high reliability and reproducibility of the proposed method. The percent recovery values were found to be between 98.70 - 101.68% with%RSD below than 1.62% for human plasma and 99.14 - 101.78% with%RSD below than 2.44% for urine samples.

Comparing analytical methods to detect SARS-CoV-2 in wastewater

Wastewater based epidemiology (WBE) has emerged as a reliable strategy to assess the coronavirus disease 2019 (COVID-19) pandemic. Recent publications suggest that SARS-CoV-2 detection in wastewater is technically feasible; however, many different protocols are available and most of the methods applied have not been properly validated. To this end, different procedures to concentrate and extract inactivated SARS-CoV-2 and surrogates were initially evaluated. Urban wastewater seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV) was used to test the concentration efficiency of an aluminum-based adsorption-precipitation method and a polyethylene glycol (PEG) precipitation protocol. Moreover, two different RNA extraction methods were compared in this study: a commercial manual spin column centrifugation kit and an automated protocol based on magnetic silica beads. Overall, the evaluated concentration methods did not impact the recovery of gamma-irradiated SARS-CoV-2 nor MgV, while extraction methods showed significant differences for PEDV. Mean recovery rates of 42.9 ± 9.5%, 27.5 ± 14.3% and 9.0 ± 2.2% were obtained for gamma-irradiated SARS-CoV-2, PEDV and MgV, respectively. Limits of detection (LoD95%) for five genomic SARS-CoV-2 targets (N1, N2, gene E, IP2 and IP4) ranged from 1.56 log genome equivalents (ge)/mL (N1) to 2.22 log ge/mL (IP4) when automated system was used; while values ranging between 2.08 (N1) and 2.34 (E) log ge/mL were observed when using column-based extraction method. Different targets were also evaluated in naturally contaminated wastewater samples with 91.2%, 85.3%, 70.6%, 79.4% and 73.5% positivity, for N1, N2, E, IP2 and IP4, respectively. Our benchmarked comparison study suggests that the aluminum precipitation method coupled with the automated nucleic extraction represents a method of acceptable sensitivity to provide readily results of interest for SARS-CoV-2 WBE surveillance.

Scaled Isolation of Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles.

Mesenchymal stem/stromal cells (MSCs) provide therapeutic effects in many diseases. Contrary to initial hypotheses, they act in a paracrine rather than a cellular manner. To this end, extracellular vesicles (EVs) have been found to mediate the therapeutic effects, even when harvested from MSC-conditioned cell culture supernatants. Lacking self-replicating activity and being so small that MSC-EV preparations can be sterilized by filtration, EVs provide several advantages as therapeutic agents over cellular therapeutics. At present, methods allowing EV preparation from larger volumes are scarce and regularly require special equipment. We have developed a polyethylene glycol-based precipitation protocol allowing extraction of EVs from several liters of conditioned medium. MSC-EVs prepared with this method have been successfully applied to a human graft-versus-host disease patient and to several animal models. Although the method comes with its own limitations, it is extremely helpful for the initial evaluation of EV-based therapeutic approaches. Here, we introduce the technique in detail and discuss all critical steps. © 2020 The Authors. Basic Protocol 1: Preparation of MSC-conditioned medium for scaled MSC-EV production Basic Protocol 2: PEG precipitation OF MSC-EV from MSC-conditioned medium.

A liquid chromatography-tandem mass spectrometry platform for the routine therapeutic drug monitoring of 14 antibiotics: Application to critically ill pediatric patients

BackgroundThe accurate measurement of plasma levels of antibiotics is crucial for the individualization of antimicrobial therapies based on PK/PD strategies. In this paper we describe a new rapid and simple LC–MS/MS platform for quantifying 14 antibiotics (amikacin, amoxicillin, ceftazidime, ciprofloxacin, colistin, daptomycin, gentamicin, linezolid, meropenem, piperacillin, teicoplanin, tigecycline, tobramycin and vancomycin) and a beta-lactamase inhibitor (tazobactam) starting from 50 μL plasma samples. MethodsAnalyses were performed on a Thermo Scientific™ Ultimate™ 3000 LC system (Thermo Fisher Scientific, Milan, Italy) coupled to a Thermo Scientific™ TSQ Quantiva™ Triple Quadrupole mass spectrometer. After fast protein precipitation protocols and addition of deuterated internal standards, samples were subjected to a fast HPLC gradient separation and the 15 drugs were quantified using multiple reaction monitoring of specific transitions over a wide range of concentrations. The suitability of the assay for TDM was tested on plasma samples derived from pediatric patients under treatment with one or more antibiotics. ResultsThe overall turnaround time of the assay was 20 min. The assay was validated following EMA guidelines for bioanalytical method validation and showed excellent accuracy (ranging from 85.3 and 112.7) and reproducibility (ranging from 1.3 to 9.7) as well as the absence of matrix effects (<15 %) for all the drugs tested. The lower limits of quantifications were between 0.1 and 2 mg/L. the recovery rate exceeded 85 % for all the drug tested. Stability was evaluated in different conditions thus allowing the setting up of reliable operative procedures. ConclusionsThis work provides a LC–MS/MS platform validated for clinical use for a rapid quantification of a broad spectrum of drugs having different chemical characteristics in a small volume of plasma and is suitable for real-time TDM-guided personalization of antimicrobial treatment in critically ill patients.

Optimisation of microbially induced calcite precipitation protocol against erosion

Five strategies of microbially induced calcite precipitation (MICP) are suggested to improve the uniformity of treatment and enhance the erosional/hydraulic response of internally unstable and poorly graded soils. The treatment consisted of the injection of a bacterial suspension and a cementation solution. The volume of the injected bacterial suspension varied from 0·3 to one pore volume, while the concentration of the cementation solution (urea/calcium chloride (CaCl2)) varied from 0·35 to 0·75 M. The biotreatment of sand columns (ϕ = 160 mm and h = 100 mm) was conducted in either one, two or three stages, with or without a low-salinity fixation solution. The biotreated specimens were then submitted to an erosion test, and the results show that alternating injection of reagents in three stages enhances the uniformity of biomineralisation, as the hydraulic conductivity is constant from the top to the bottom of the specimens. This protocol also prevents internal erosion as the critical hydraulic gradient (i cr) is equal to 10 and the cumulative fine loss (M e) does not exceed 63 g/m2. Finally, the use of a fixation solution prevents any clogging near the injection point and stimulates the bacterial transport in the soil.

Anion exchange resin and slow precipitation preclude the need for pretreatments in silver phosphate preparation for oxygen isotope analysis of bioapatites

Preventing the inclusion of oxygen bearing compounds from the organic fraction of skeletal tissues is often considered key to obtaining faithful δ18O measurements of the mineral fraction, which are widely used across the archaeological, forensic and geochemical sciences. Here we re-explore the contentious issue of organic removal pretreatments by establishing how different silver phosphate preparation methods perform in producing pure silver phosphates with a faithful biogenic isotopic signal. We then compare this baseline performance to a pretreatment based approach. Our results show that anion exchange purification combined with slow precipitation of silver phosphate consistently produces silver phosphates of high purity without prior pretreatment. Rapid precipitation protocols without additional purification, while effective and time-efficient for low organic samples such as enamel, suffer from the inclusion of substantial amount of organic matter in silver phosphates from bone or dentine samples. However, despite substantial organic contamination in such samples, δ18O values do not necessarily show substantial shifts. Further study is needed to clarify the reason for this, but for now the use of an anion exchange based protocol represents the most cautious approach to processing bone and dentine samples and we recommend its use for such samples. Confirming previous work we find H2O2 pretreatment to be only partially effective at removing higher amounts of organic matter. Both H2O2 and NaOCl pretreatments show unpredictable side effects on δ18O values of both bones and inorganic samples. We additionally find no indication that the presence of organic material hinders the dissolution of bioapatite samples.