Optimized Solid-phase Extraction Method Research Articles

Optimization of reversed-phase solid-phase extraction for shotgun proteomics analysis.

Reversed-phase solid-phase extraction (SPE) is the method of choice for the purification of proteomics samples. Even though the efficacy of SPE methods is sample type-dependent, the manufacturers' protocols are used in most studies. Using an optimized SPE method can lead to a substantial gain in identification and recovery. In this tutorial, we give a brief introduction to the most important parameters influencing SPE performance, and we present a short workflow (16 measurements) for optimizing the SPE procedure. This is complemented by method performance assessment instructions and a short troubleshooting guide to help users further understand and investigate their SPE methods.

Simultaneous monitoring of multiple hormones from human islets of Langerhans using solid-phase extraction-mass spectrometry.

Islets of Langerhans release peptide hormones in controlled amounts and patterns to ensure proper maintenance of blood glucose levels. The overall release of the hormones is shaped by external factors and by autocrine and paracrine interactions occurring within the islets. To better understand what controls the secretion of islet-secreted peptides, and how these processes go awry in diabetes, methods to monitor the release of multiple hormones simultaneously are needed. While antibody-based assays are typically used, they are most often applied to quantification of a single hormone. Mass spectrometry (MS), on the other hand, is well suited for quantifying multiple hormones simultaneously but typically requires time-consuming separation steps with biological samples. In this report, response surface methodology was used to identify a set of optimal solid-phase extraction (SPE) conditions for the islet-secreted peptides, insulin, C-peptide, glucagon, and somatostatin. The optimized SPE method was used with multiple reaction monitoring and isotopically labeled standards to quantify secretion levels. Calibrations were linear from 0.5 to 50 nM with <15% RSD peak area ratios. A microfluidic system was used to perfuse 30 human islets with different glucose conditions, and fractions were collected every 2 min for SPE-MS analysis. Results showed the release dynamics of the individual peptides, as well as patterns, such as positively and negatively correlated release and oscillations. This rapid SPE-MS method is expected to be useful for examining other peptide and small-molecule secretions from islets and could be applied to a number of other biological systems for investigating cellular communication.

Solid-Phase extraction of polycyclic aromatic hydrocarbons from water samples using CTAB-TIO2 modified nanotubes

To extract polyaromatic hydrocarbons (PAHs) from water samples using the Solid Phase Extraction (SPE) method. Based on hemimicelle/admicelle formation on the oxide surface, the cetyltrimethylammonium bromide-titanate nanotube system has been used as an adsorbent for PAHs extraction. Many experiments were conducted to optimize the extraction parameters, and resulting extracts were analyzed using Gas Chromatography-Flame Ionization Detection. Results showed that the kind and volume of elution solvent, the pH, volume, and concentrations of the sample, and the pH PAH and concentration of the cetyltrimethylammonium bromide significantly affect extraction yields. Under optimal conditions, modified TiO2 nanotubes exhibited their good enrichment capacity for PAHs (∼100%). Furthermore, the method of the analysis showed good linearity (R2 greater than 0.99), satisfactory repeatability (Relative standard deviation RSD between 4.8 and 11.2), and detection limits between 0.08 and 0.3 ng mL-1. The present SPE method showed competitive results with those given using multiwalled carbon nanotubes as adsorbent, and even better than those determined by the usual SPE-C18-silica method and liquid–liquid – extraction. The proposed method was applied to real water samples, and recoveries were between 80 and 99%, showing minimal matrix effect and the robustness of the optimized SPE method.

Profiling milk oligosaccharides in Philippine buffalo breeds across lactation and identification of novel high-molecular weight fucosylated and sialylated oligosaccharides

Animal milk oligosaccharides have attracted considerable interest because some sources can mimic the diversity of oligosaccharides found in human milk, associated with many health benefits. This study is the first to characterize buffalo milk oligosaccharides across lactation from representative Philippine breeds and crosses: traditionally draft-purpose Philippine Carabao, dairy-type Bulgarian Murrah, and their 50%, 75%, 87%, 93% crossbreeds. An optimized solid-phase extraction method combined with nano-LC-chip/QTOF mass spectrometry was developed, generating a bioinformatic library of 66 oligosaccharides. Fourteen fucosylated and 9 fucosylated-sialylated oligosaccharides were identified, with 22 novel oligosaccharides not previously found in human or bovine milk. The 93% crossbreed and Philippine Carabao breed had higher numbers of fucosylated oligosaccharides and novel oligosaccharides overall. The 93% crossbreed had higher diversity and total and sialylated oligosaccharides abundances across lactation stages. This is the most comprehensive study on buffalo milk oligosaccharides to date and indicates a strong potential for use in infant formulas.

Broad-range extraction of highly polar to non-polar organic contaminants for inclusive target analysis and suspect screening of environmental samples

The lack of carefully optimized extraction techniques for the analysis of compounds with diverse polarities limits the identification of toxic pollutants in aqueous environmental matrices, particularly those that are considered persistent and mobile organic compounds (PMOCs). Tailored extraction techniques for specific classes of chemicals often result in very low to no extraction of either very polar or relatively non-polar chemicals, depending on the sorbent used. Hence, it is crucial to develop a balanced extraction for a wider range of polarity, especially for non-target analysis of chemical residues, in order to capture the occurrence of the full profile of micropollutants. Herein, a tandem solid-phase extraction (SPE) technique incorporating both hydrophilic-lipophilic balance (HLB) and mixed-mode cation exchange (MCX) sorbents was developed to extract and analyze 60 model compounds with a wide range of polarities (log Kow from ‐1.9 to 5.5) from untreated sewage matrices. Extraction efficiencies were assessed in NanoPure™ water and untreated sewage samples; 51 compounds in NanoPure™ water and 44 compounds in untreated sewage had ≥60 % extraction recoveries using the developed tandem SPE method. The method limits of detection ranged from 0.25 to 88 ng/L in untreated sewage matrix. The applicability of the extraction method was demonstrated in untreated wastewater samples; using the tandem SPE for suspect screening analysis captured an additional 22 compounds that were not extracted when using the HLB sorbent only. The optimized SPE method was also evaluated for the extraction of per- and polyfluoroalkyl substances (PFAS) by analyzing the same sample extracts under negative electrospray ionization liquid chromatography-tandem mass spectrometry (LC-MS/MS). The wastewater samples revealed the presence of sulfonamide-, sulfonic-, carboxylic-, and fluorotelomer sulfonic- PFAS with chain lengths 8, 4–8, 4–9, and 8, respectively, indicating that the tandem SPE procedure provides an efficient one-step extraction for the analysis of PMOCs that include pharmaceuticals, pesticides, and PFAS.

Testing Clean-Up Methods for the Quantification of Monosaccharides and Uronic Acids

The determination of carbohydrate composition is extremely important for quality control in food and beverages, in material science, in pharmaceutics, and in the field of cultural heritage. Considering the complexity and the heterogeneity of the matrices, the optimization of extraction and purification steps aiming at maximizing the saccharide recovery from the matrix and effectively removing interferences is mandatory. The presence of inorganic components, besides being detrimental to the analytical instrumentation, can catalyze the isomerization of some sugars causing an alteration to their quantitative and qualitative profiles. In the present paper, protocols for suppressing the interference of inorganic ions in the quantification of monosaccharides and uronic acids by Gas Chromatography–Mass Spectrometry (GC-MS) are proposed. Two clean-up methods based on ion exchange resins (Amberlite MB-6113 and Amberlite IRN-150) and one making use of solid-phase extraction with a polypropylene Solid-Phase Extraction (SPE) column were tested on a standard carbohydrate solution, and the elution conditions optimized. The best purification conditions, in terms of higher recovery yield values for seven monosaccharides and two uronic acids, were obtained using SPE. Furthermore, the optimized SPE method was validated on a sample of mural painting rich in saccharides and inorganic material.

Development of a Solid Phase Extraction-Based Method for the Quantitative Analysis of Methylmercury in Soil and Sediment.

A simple, universal method to quantify soil methylmercury (MeHg) is not available. Here, we developed a solid-phase extraction-based method using gas chromatography mass spectrometry. MeHg was purified from the soil matrix using an optimized solid-phase extraction method, which reduced the use of organic solvents and eliminated the requirement for harmful reagents. The sample limit of quantification was 7.5ng/g. MeHg recovery in the reference samples was 96.2%-102.6%; the intra- and inter-assay coefficients of variation were 3.4%-7.1% and 4.3%-7.1%, respectively, indicating high validation performance. This analysis method is simple as it can be performed using general-purpose reagents and instruments; has a high degree of trueness and accuracy; could be useful for soil MeHg quantification with improved sensitivity; and can provide reliable data to prevent MeHg contamination and improve food safety.

LC-MS/MS determination of glimepiride in human plasma with a high recovery at picogram scale: Pharmacokinetic study after oral administration

Abstract Although glimepiride (GLM) is the first-line treatment of Type II diabetes, low extraction recovery is still a significant limitation in previous plasma analysis methods. An optimized solid-phase extraction method of GLM in human plasma with excellent extraction recovery, 100 ± 0.06%, was achieved using liquid chromatography-electrospray ionization tandem mass spectrometry and Gliclazide (GLZ) as an internal standard. GLM was extracted from 100 µL plasma sample using Sep-Pak® vac 1cc (100 mg) C18 column and methylene chloride: methanol (2: 1, v/v) as eluant. Both GLM and GLZ were monitored by a triple quad mass spectrometer applying positive multiple reaction monitoring mode (+MRM). The protonated precursor ions and product ions of GLM and GLZ were m/z 491(352), and m/z 324 (127), respectively. The detection and measurement of low levels of GLM in human plasma reached to picogram range (limit of detection (LOD) = 60 pg/mL, limit of quantification (LOQ) = 200 pg/mL). The method was validated in terms of selectivity, linearity, recovery, accuracy, and precision. The method was successfully applied to the pharmacokinetic study of GLM following oral administration of 1 mg GLM tablets to 12 healthy volunteers.

Application of amino-based chitosan cyclodextrin derivatives for the extraction of catechins in green tea with high-performance liquid chromatography.

Chitosan derivatives have been studied widely, but poor solubility in water restricts their applications. In this study, four types of amine-based chitosan derivatives were prepared and modified further with beta-cyclodextrin. The sequential microextraction of catechins ((+)-catechin and (-)-epigallocatechin gallate) from green tea powder by an optimized solid-phase extraction method using these four derivatives was investigated. The optimal conditions for the extraction of catechins were 60°C for a 40min extraction period. The purity and amount of each catechin were determined by high-performance liquid chromatography. The different amines strengthened the extraction capacity of chitosan. Among the four types of amines, ethylene diamine grafted chitosan beta-cyclodextrin had the highest extraction capacity to catechins. Therefore, this material was used in the extraction assay, and the standard curves of (+)-catechin and (-)-epigallocatechin gallate were linear over the concentration range, 0.25-500µg/mL, after assaying five data points in duplicate. Solid-phase extraction with the amino-based chitosan beta-cyclodextrin system is a new application of chitosan, which has potential applications in the extraction of bioactive compounds from plant materials or the removal of different impurities from specific extracts.

Tetrafluoroterephthalonitrile-crosslinked β-cyclodextrin polymers for efficient extraction and recovery of organic micropollutants from water

In this study, we evaluated the performance of a novel tetrafluoroterephthalonitrile-crosslinked β-cyclodextrin polymer (TFN-CDP) as a solid-phase extraction (SPE) material for the recovery of up to 189 diverse organic micropollutants (MPs) from water. The optimized extraction procedure requires loading of water samples adjusted to a pH of 3 onto 500 mg of TFN-CDP packed into an SPE cartridge. Under these conditions, 88.7% of the MPs have average extraction efficiencies greater than 80%. The optimized recovery procedure requires elution with 15 mL of methanol amended with 15 mg of calcium chloride. Under these conditions, 58.4% of the MPs have average absolute recoveries between 80% and 120%. We compared the performance of the optimized SPE method for TFN-CDP with a previously optimized SPE method employing hydrophilic-lipophilic balance (HLB) adsorbents in nanopure water and in wastewater-impacted surface water. The data indicate that the optimized TFN-CDP method performs as well as or better than the optimized HLB-based SPE method. These findings represent an important step forward in the development of sustainable and inexpensive materials for the extraction and recovery of organic MPs from water.

Optimization of the Determination Method for Dissolved Cyanobacterial Toxin BMAA in Natural Water.

There is a serious dispute on the existence of β-N-methylamino-l-alanine (BMAA) in water, which is a neurotoxin that may cause amyotrophic lateral sclerosis/Parkinson's disease (ALS/PDC) and Alzheimer' disease. It is believed that a reliable and sensitive analytical method for the determination of BMAA is urgently required to resolve this dispute. In the present study, the solid phase extraction (SPE) procedure and the analytical method for dissolved BMAA in water were investigated and optimized. The results showed both derivatized and underivatized methods were qualified for the measurement of BMAA and its isomer in natural water, and the limit of detection and the precision of the two methods were comparable. Cartridge characteristics and SPE conditions could greatly affect the SPE performance, and the competition of natural organic matter is the primary factor causing the low recovery of BMAA, which was reduced from approximately 90% in pure water to 38.11% in natural water. The optimized SPE method for BMAA was a combination of rinsed SPE cartridges, controlled loading/elution rates and elution solution, evaporation at 55 °C, reconstitution of a solution mixture, and filtration by polyvinylidene fluoride membrane. This optimized method achieved > 88% recovery of BMAA in both algal solution and river water. The developed method can provide an efficient way to evaluate the actual concentration levels of BMAA in actual water environments and drinking water systems.

Isolation and structural elucidation by 2D NMR of planteose, a major oligosaccharide in the mucilage of chia (Salvia hispanica L.) seeds

An oligosaccharide was isolated in high purity and excellent yield from the water-extractable mucilage of chia (Salvia hispanica L.) seeds using an optimized solid-phase extraction method. LC–MS analysis showed that the compound presents a molecular mass of 504Da and trifluoroacetic acid hydrolysis revealed that it consists of galactose, glucose and fructose. Glycosidic linkage analysis showed that the oligosaccharide contains two non-reducing ends corresponding to terminal glucopyranose and terminal galactopyranose, respectively. The oligosaccharide was identified as planteose by the complete assignment of a series of 2D NMR spectra (COSY, TOCSY, ROESY, HSQC, and HMBC). The significance of the presence of planteose in chia seeds is discussed in the context of nutrition and food applications.

Development of a Rapid LC-MS/MS Method for the Determination of Emerging Fusarium mycotoxins Enniatins and Beauvericin in Human Biological Fluids.

A novel method for the simultaneous determination of enniatins A, A1, B and B1 and beauvericin, both in human urine and plasma samples, was developed and validated. The method consisted of a simple and easy pretreatment, specific for each matrix, followed by solid phase extraction (SPE) and detection by high performance liquid chromatography-tandem mass spectrometry with an electrospray ion source. The optimized SPE method was performed on graphitized carbon black cartridges after suitable dilution of the extracts, which allowed high mycotoxin absolute recoveries (76%–103%) and the removal of the major interferences from the matrix. The method was extensively evaluated for plasma and urine samples separately, providing satisfactory results in terms of linearity (R2 of 0.991–0.999), process efficiency (>81%), trueness (recoveries between 85% and 120%), intra-day precision (relative standard deviation, RSD < 18%), inter-day precision (RSD < 21%) and method quantification limits (ranging between 20 ng·L−1 and 40 ng·L−1 in plasma and between 5 ng·L−1 and 20 ng·L−1 in urine). Finally, the highly sensitive validated method was applied to some urine and plasma samples from different donors.

Determination of phthalate esters in non-alcoholic beverages by GC–MS and optimization of the extraction conditions

A quantificational method for 7 phthalate esters in non-alcoholic beverages was developed. Dimethyl phthalate, di-ethyl phthalate, di-propyl phthalate (DPP), di-butyl phthalate (DBP), benzyl butyl phthalate, di-(2-ethylhexyl) phthalate (DEHP), and di-octyl phthalate (DOP) were extracted from non-alcoholic beverages with the optimized solid-phase extraction method, and quantification was achieved by gas chromatography–mass spectrometry with isotope internal standard of d4-di-(2-ethylhexyl) phthalate (DEHP-d4). The inter-day method repeatability (RSD) was 8–13 %, whereas the intra-day method repeatability (RSD) was 9–15 %. The mean spiking recoveries were 84–105 %. A wide variety of phthalate concentrations was observed in 48 non-alcoholic beverages. DEHP was the most abundant phthalate compound followed by DBP, DPP, and DOP. DEHP was found in sport drinks (0.015–0.098 mg L−1), tea (0.016–0.123 mg L−1), coffee (0.028–0.159 mg L−1), and fruit juices (0.022–0.126 mg L−1).

Optimized extraction method for LC–MS determination of bisphenol A, melamine and di(2-ethylhexyl) phthalate in selected soft drinks, syringes, and milk powder

Described below is an optimized solid-phase extraction method (SPE) for the simultaneous determination of three hazardous plastic additives. Two high-performance liquid chromatographic-electrospray ionization-mass spectrometric (HPLC-ESI-MS) methods were developed and validated to estimate the melamine (MEL), bis(2-ethylhexyl) phthalate (DEHP), and bisphenol A (BPA) contents in drinking water, syringes, soft drinks, and dry milk powder. One extraction procedure optimally recovered all three substances from the different matrices. Two extraction columns were combined and included a silica gel LiChroprep RP-2 column (20:1, g/g, top column) and a Sep-Pak with a C18 column (500mg, bottom column). The analytical column was an Agilent Eclipse XDB-C8 column, 4.6mm×150mm, 5μm, maintained at 50±2°C. MEL and DEHP were monitored by positive triple quad mass spectrometry (TQ-MS) using an acidic mobile phase, while BPA was monitored by negative TQ-MS using a mobile phase containing a 0.05% ammonia solution. The general linear range of the three compounds ranged from 12 to 1000pg/μL in the injected solution (25μL). The average extraction recoveries were within the range of 83.0-102.5%. Relatively high concentrations of BPA and DEHP were found in the milk powder and sterile syringes.

Simultaneous Determination of 4-Substituted Cathinones (4-MMC, 4-MEC and 4-FMC) in Human Urine by HPLC-DAD

This study developed a selective and rapid high-performance liquid chromatography-diode array detection method for the confirmation of different cathinone derivates in human urine. Samples were prepared by solid-phase extraction (SPE) using procaine hydrochloride as the internal standard. The chromatographic separation was performed on a Kinetex PFP column using isocratic elution. The mobile phase was composed of a mixture of acetonitrile (33%, v/v) and 0.005M ammonium trifluoroacetate buffer (67%, v/v; pH 4.93 ± 0.03) with a flow rate of 0.350 mL/min. The diode array detection was performed at 262 nm. The method was linear over the concentration range of 25-2,400 ng/mL. Intra-day and inter-day precision values for cathinones were less than 1.26% (relative standard deviation). The limit of detection for any compounds extracted from human urine by the optimized SPE method was 40 ng/mL and the limit of quantification was 100 ng/mL in the urine. The recovery rate of SPE was between 71 and 82% with a lower relative standard deviation than 2.35%.

Multireaction monitoring of 12 peptides for lowered immunity screening

A multireaction monitoring method using high-performance liquid chromatography-tandem mass spectrometry was developed for 12 target peptides for determination of endogenous peptide concentrations in human serum. Chromatographic separation conditions were optimized and recoveries for liquid-liquid extraction, solid-phase extraction (SPE), and ultrafiltration of endogenous peptides from human serum were compared, and the SPE method was selected for 12 targeted peptide extractions. The optimized SPE method gave recoveries higher than 60 % for all targeted peptides. The limit of detection was 10 ng/ml for most peptides, except for N-formyl-methionyl-leucyl-phenylalanine (NFMLP) and adrenocorticotropic hormone (ACTH) (18-39). The limit of detection for these two peptides was 1 ng/ml. The real serum samples of 25 elderly and 23 young people were analyzed using the optimized extraction and analysis method. Half of the 12 peptides were below the limit of quantification, and B-type natriuretic peptide, cholecystokinin, ACTH(7-38), substance P, NFMLP, and valyl-glutamyl-prolyl-isoleucyl-prolyl-tyrosine were quantified in the concentration range from 0.1 to 50 ng/ml. The concentration of ACTH(7-38) was significantly higher in elderly people and that of NFMLP was significantly lower in elderly people compared with young people (p < 0.0001). This result implies that there be a possible relationships between ACTH, NFMLP and lowered immunity.

Solid-phase extraction of carotenoids

In this work, solid-phase extraction (SPE) trapping performance of lutein and β-carotene, which were used as the model molecules of carotenoids, was investigated. The absorption, elution, and enrichment of carotenoids on SPE cartridges with four different sorbents, i.e. C 30, C 18, diol, and silica, were compared respectively with the help of frontal analysis technique. The high retentions of both lutein and β-carotene were achieved on the C 18 and C 30 cartridges. The diol and silica cartridges only had good retention for lutein. The optimized SPE method for sample pretreatment for the carotenoids analysis was obtained after the investigation of trapping performance. The method was applied successfully to the analysis of biological sample, i.e. serum and human breast milk. The recovery, accuracy, and precision of SPE method comparing with those of traditional liquid–liquid extraction (LLE) method for the sample pretreatment for the analysis of carotenoids owned a number of advantages such as rapid, no chloroform used, and accurate versus LLE.

Functional Characterization of the Flagellar Glycosylation Locus in Campylobacter jejuni 81–176 Using a Focused Metabolomics Approach

Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81-176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81-176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-L-glycero-alpha-L-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways.

Isolation of compounds from bleached kraft mill recovery condensates associated with reduced levels of testosterone in mummichog (Fundulus heteroclitus)

Previous studies have identified chemical recovery condensates as a primary source of hormonally active substances within a Canadian bleached kraft pulp mill. Although reverse osmosis treatment of condensates raises the exposure threshold that alters circulating levels of testosterone in mummichog (Fundulus heteroclitus), the responsible chemicals have not been characterized. In this study, a solid-phase extraction (SPE) method was developed to isolate chemical recovery condensate extractives for evaluation of hormonal activity. Condensates generated during softwood and hardwood pulp production were investigated for their relative potential to affect both circulating and gonadal production of sex steroids in mummichog. Mummichog were exposed to whole condensates, extracts from suspended particulates (> 1 microm), two fractions from SPE, and residual condensates after SPE. The distribution of bioactivity among condensate fractions was similar for both wood furnishes. In both sexes, significant depressions in circulating testosterone and in in vitro gonadal testosterone production were associated with exposure to whole condensates, particulate extracts, one SPE fraction, and residual material after SPE. An optimized SPE method subsequently demonstrated complete recovery of polar, bioavailable chemicals that reduced testosterone levels in both sexes. Characterizations of active fractions by gas chromatography-mass spectrometry (GC-MS) showed the presence of extractives with molecular masses > or = 240 amu possessing functionalities consistent with lignin degradation products. This study provides the first isolation of chemicals derived from pulp production associated with impaired reproductive performance in fish.