Two-step Solid-phase Extraction Research Articles

Quantitation of total vitamin D2 and D4 in UV-exposed mushrooms using HPLC with UV detection after novel two-step solid phase extraction

A robust method for quantitation of total vitamin D2 and D4 in mushrooms by high performance liquid chromatography with UV detection (HPLC-UV) was developed to analyze mushrooms exposed to UV light. A two-step solid phase extraction (SPE) (silica, carbon black) removed chromatographic interferences typically resolved only with mass spectrometric detection (LC-MS) and allowed quantitation of all vitamin D and pre-D analytes. The vitamin and pre-vitamin forms of D2, D4 and D3 (internal standard), as well as other photoisomers and sterols were resolved. Results for six types of UV-exposed mushrooms were comparable to LC-MS. Screening of ten additional types of UV-exposed mushrooms without the IS confirmed lack of interference with the IS. The limit of quantification (µg/100 g fresh weight) was 0.4 for vitamin D and 0.9 for pre-vitamin D. Mushrooms do not have to be dried, and separatory funnels and large solvent volumes were also eliminated from sample preparation.

Validation of an isotope dilution mass spectrometry (IDMS) measurement procedure for the reliable quantification of steroid hormones in waters.

Reliable data are compulsory to efficiently monitor pollutants in aquatic environments, particularly steroid hormones that can exert harmful effects at challenging analytical levels below the ng L-1. An isotope dilution two-step solid-phase extraction followed by an ultra-performance liquid chromatography separation coupled to tandem mass spectrometry (UPLC-MS/MS) detection method was validated for the quantification of 21 steroid hormones (androgens, estrogens, glucocorticoids, and progestogens) in whole waters. To achieve a realistic and robust assessment of the performances of this method, the validation procedure was conducted using several water samples representative of its intended application. These samples were characterized in terms of concentration of ionic constituents, suspended particulate matter (SPM), and dissolved organic carbon contents (DOC). For estrogens that are part of the European Water Framework Directive Watchlist (17beta-estradiol and estrone), the performances met the European requirements (decision 2015/495/EU) in terms of limit of quantification (LQ) and measurement uncertainty. For 17alpha-ethinylestradiol, the challenging LQ of 0.035ng L-1 was reached. More generally, for 15 compounds out of 21, the accuracy, evaluated in intermediate precision conditions at concentrations ranging between 0.1 and 10ng L-1, was found to be within a 35% tolerance. The evaluation of the measurement uncertainty was realized following the Guide to the expression of Uncertainty in Measurement. Finally, a water monitoring survey demonstrated the suitability of the method and pointed out the contamination of Belgium rivers by five estrogens (17alpha-ethinylestradiol, estriol, 17alpha-estradiol, 17beta-estradiol, and estrone) and three glucocorticoids (betamethasone, cortisol, and cortisone) which have been up to now poorly documented in European rivers.

The Isolation of Specialty Compounds from Amphidinium carterae Biomass by Two-Step Solid-Phase and Liquid-Liquid Extraction.

The two main methods for partitioning crude methanolic extract from Amphidinium carterae biomass were compared. The objective was to obtain three enriched fractions containing amphidinols (APDs), carotenoids, and fatty acids. Since the most valuable bioproducts are APDs, their recovery was the principal goal. The first method consisted of a solid-phase extraction (SPE) in reverse phase that, for the first time, was optimized to fractionate organic methanolic extracts from Amphidinium carterae biomass using reverse-phase C18 as the adsorbent. The second method consisted of a two-step liquid-liquid extraction coupled with SPE and, alternatively, with solvent partitioning. The SPE method allowed the recovery of the biologically-active fraction (containing the APDs) by eluting with methanol (MeOH): water (H2O) (80:20 v/v). Alternatively, an APD purification strategy using solvent partitioning proved to be a better approach for providing APDs in a clear-cut way. When using n-butanol, APDs were obtained at a 70% concentration (w/w), whereas for the SPE method, the most concentrated fraction was only 18% (w/w). For the other fractions (carotenoids and fatty acids), a two-step liquid-liquid extraction (LLE) method coupled with the solvent partitioning method presented the best results.

Oxysterols and Oxysterol Sulfates in Alzheimer's Disease Brain and Cerebrospinal Fluid.

Background:Brain cholesterol levels are tightly regulated but increasing evidence indicates that cholesterol metabolism may drive Alzheimer’s disease (AD)-associated pathological changes. Recent advances in understanding of mitochondrial dysfunction in AD brain have presented a vital role played by mitochondria in oxysterol biosynthesis and their involvement in pathophysiology. Oxysterol accumulation in brain is controlled by various enzymatic pathways including sulfation. While research into oxysterol is under the areas of active investigation, there is less evidence for oxysterol sulfate levels in human brain.Objective:This study investigates the hypothesis that AD brain oxysterol detoxification via sulfation is impaired in later stages of disease resulting in oxysterol accumulation.Methods:Lipids were extracted from postmortem frozen brain tissue and cerebrospinal (CSF) from late- (Braak stage III-IV) and early- (Braak stage I-II) stage AD patients. Samples were spiked with internal standards prior to lipid extraction. Oxysterols were enriched with a two-step solid phase extraction using a polymeric SPE column and further separation was achieved by LC-MS/MS.Results:Oxysterols, 26-hydroxycholesterol (26-OHC), 25-hydroxycholesterol (25-OHC), and 7-oxycholesterol levels were higher in brain tissue and mitochondria extracted from late-stage AD brain tissue except for 24S-hydroxycholesterol, which was decreased in late AD. However, oxysterol sulfates are significantly lower in the AD frontal cortex. Oxysterols, 25-OHC, and 7-oxocholesterol was higher is CSF but 26-OHC and oxysterol sulfate levels were not changed.Conclusion:Our results show oxysterol metabolism is altered in AD brain mitochondria, favoring synthesis of 26-OHC, 25-OHC, and 7-oxocholesterol, and this may influence brain mitochondrial function and acceleration of the disease.

Determination of Aminophosphonate Herbicides in Glutamate Loaded Spice Mix by LC-IDMS and Method Extension to Other Food Matrices

The accumulation of organophosphorus type herbicides has been observed worldwide in the environment (i.e. soil, water), together with their appearance in foods of plant origin. This paper reports a new liquid chromatography–isotope dilution–tandem mass spectrometric method (LC-IDMS) for the analysis of glufosinate (GLUF), glyphosate (GLY) and its main metabolite, aminomethylphosphonic acid (AMPA), in challenging food samples. Sample preparation is based on aqueous extraction with ethylenediaminetetraacetic acid solution, followed by solid-phase extraction (SPE) on mixed-mode cation exchange cartridges to remove matrix constituents before derivatization with 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl). Derivatized samples were cleaned up on hydrophilic modified polymeric SPE cartridge. This two-step SPE supported sample preparation approach, and the LC-IDMS separation carried out in negative ionization mode resulted in fit-for-purpose recovery (81–118%) and precision (4–18%) in the validation of glutamate loaded spice mix, mushroom, maize and cherry samples. Amino acid content influencing FMOC derivatization efficiency was estimated with a HILIC-MS/MS setup. Multiple reaction monitoring (MRM) was assisted with high-resolution (QTOF) accurate mass data on the FMOC-derivatized GLUF, GLY and AMPA standards. The limit of quantification (LOQ) was 0.005 mg/kg for all the three analytes. The method was successfully applied on quality control samples (oat and arugula) with fit-for-purpose accuracy (99–120%) and on other nineteen real samples, where GLY and AMPA were detected in the range between 0.005 and 0.069 mg/kg.

Detection of Ustiloxin A in urine by ultra-high-performance liquid chromatography-tandem mass spectrometry coupled with two-step solid-phase extraction

Due to global outbreak of rice false smut disease, ustiloxin A (UA) was detected in rice. However, accurate methods for monitoring UA in human body fluids were lacking. In this context, a UPLC-MS/MS method based on two-step SPE was constructed for measuring UA in urine. The limits of UA quantification in human and mice urine were 58.3 and 108.7 ng/L, respectively. The proposed method was applied to detect UA in urine samples collected from human and mice. After dietary exposure, the contents of UA in mice urine were from 6.03 to 16.76 μg/g of creatine, accounting for approximate 14% of daily intake dose. Furthermore, due to the trace residues in rice (78–109 ng/kg), no detectable UA was observed in the urine of 20 volunteers. To the best of our knowledge, it is the first time to report the occurrence of UA in mammal urine.

Development of a GC-FID method for the quantitative determination of polyglycerol polyricinoleate (PGPR) in foods

ABSTRACT Polyglycerol polyricinoleate (PGPR) is a powerful lipophilic emulsifier used in low-fat spreads and chocolate. It should be used at the lowest level at which the desired technological effect is achieved, not exceeding the specified maxima according to Annexe II to Regulation (EC) No 1333/2008. A gas chromatography–flame ionisation detection (GC-FID) method was developed for quantification of PGPR. This method is based on estimating the content of ricinoleic acid using 12-hydroxyoctadecanoic acid as an internal standard, from which the PGPR concentration was deduced. The method involved saponification, methylation, a two-step solid phase extraction (SPE) separation of the fatty acid methyl esters (FAMEs), silylation, and GC-FID analysis. The limits of detection and quantification of ricinoleic acid were 2.2 and 6.7 μg/mL, respectively, at 0.1 µL injection volume. Considering the average content of ricinoleic acid in PGPR (i.e. 86.63 ± 2.0 wt%) and the amount of food product that is used in the proposed protocol (i.e. 20 mg), this resulted in a LOD and LOQ of 0.76 and 2.32 μg PGPR per mg of food product, respectively. The developed method was validated by determining PGPR recovery from a high oleic sunflower oil (HOSO) solution, from chocolate spiked with a commercially available PGPR, and from commercially available low fat spreads with a known PGPR content. The actual recovery was more than 95% for all matrices, indicating the accuracy of the developed analytical technique. Moreover, the method proved to be very reproducible, with RSD < 4% for concentrations ranging from 0.2 to 5 wt%. The results showed that our proposed GC-FID method enables the reliable and quantitative determination of the PGPR concentration in commercial food products with various fat contents.

Development and implementation of an analytical procedure for the quantification of natural and synthetic steroid hormones in whole surface waters

Natural and synthetic steroid hormones are chronically released into aquatic spheres. Whereas knowledge on their combined mode of action and the cocktail effect are needed, only few multi-class methods address the challenge of their trace quantification in surface waters. The current study describes a sensitive multi-residue analytical strategy aiming to quantify 23 steroid hormones belonging to androgens, estrogens, glucocorticoids and progestogens in whole surface waters. The procedure relies on a two-step solid-phase extraction followed by an ultra-performance liquid chromatography separation coupled to tandem mass spectrometry detection (UPLC-MS/MS). Isotope dilution was implemented when possible in order to ensure the reliability of the measurement. The procedure was optimized toward the reliable quantification of the 23 target compounds at the predicted no-effect concentrations when existing or below the ng L−1 level. Satisfactory absolute global recoveries ≥ 77% were obtained for almost all compounds (21 out of 23) in intermediate precision conditions. Measurement errors were comprised between −27% and +17% for the great majority of compounds (21 out of 23) with standard deviations < 20% in intermediate precision conditions. Despite signal suppression was observed in water samples, satisfactory limits of quantification were achieved, ranging from 0.035 ng L−1 for 17alpha-ethinylestradiol to 1 ng L−1 for 6beta-hydroxycortisol and 6beta-hydroxydexamethasone. Abiotic stability was demonstrated for the great majority of target compounds (22 out of 23) in reference water samples stored at 4 ± 3 °C during 48 h, driving our sampling strategy. To demonstrate its fitness for purpose, the procedure was implemented in a preliminary monitoring survey of Belgian surface waters. As a result, 6 out of 23 target compounds were detected or quantified, showing a contamination by some estrogens and glucocorticoids at levels ranging from 0.1 to 0.9 ng L−1.

Uptake and release profiles of PEGylated liposomal doxorubicin nanoparticles: A comprehensive picture based on separate determination of encapsulated and total drug concentrations in tissues of tumor-bearing mice

The PEGylated liposomal nanoparticle has been widely used as a carrier in drug delivery system. To become biologically active, the encapsulated drug must be released from the nanoparticle vehicle. However, due to limitations of current bioanalytical methods, the characterization of this release process has been restricted to determination of total drug in tissues and tumor. As a result, the fate of liposomal nanoparticles including their uptake into target tissue has not been fully characterized. In this study, we developed a novel two-step solid phase extraction on two separated columns procedure to separate liposomes from tissues and tumors without liposomal leakage. This allowed us to determine encapsulated drug, total drug and, by difference, released drug and compare the release and uptake profiles of PEGylated liposomal doxorubicin in tissues and tumor of tumor-bearing mice with corresponding profiles for free doxorubicin. The liposomal nanoparticles released doxorubicin into tumor efficiently and, compared with administration of free drug, increased doxorubicin uptake into tumor by 1.8-fold. It also decreased doxorubicin uptake into heart (0.78-fold lower) with the potential to reduce doxorubicin cardiotoxicity. Drug release reached constant levels in tissues and tumor after 12 h with released doxorubicin concentration remaining at 70–80% of total doxorubicin concentration and in tumor at 86% of total drug concentration. The assay also included determination of the main doxorubicin metabolites. Determination of the metabolites showed that liposomal entrapment delays and decreases the metabolism of doxorubicin but does not alter the metabolic pathway. These results provide a clear and comprehensive picture of the biodistribution of doxorubicin administered in liposomal nanoparticles which may assist in the rational design of other liposomal nanoparticles.

Toward the determination of estrogenic compounds in the framework of EU watch list: validation and implementation of a two-step solid phase extraction–liquid phase chromatography coupled to tandem mass spectrometry method

In this paper, development and optimization of a new method for determination of the three hormones (estrone, 17-β-estradiol and 17-α-ethinylestradiol) of the Watch list were described. The validated method relies on isotope dilution—two-step solid phase extraction–liquid phase chromatography mass spectrometry method. The measurement procedure validation has been performed in real matrices, including matrix with high suspended particulate matter and high organic carbon contents, to demonstrate its fitness for purpose. Method performances were in agreement with the requirements of the decision 2015/495/EU (maximal acceptable method detection limit and whole-water analysis). Limits of quantification of the method were of 0.4 ng L−1 for estrone and 17-β-estradiol and 0.1 ng L−1 for 17-α-ethinylestradiol. Expanded uncertainties (k = 2) at limit of quantification were equal to 35 % for estrone and 17-β-estradiol and 50 % for 17-α-ethinylestradiol. The method was successfully implemented to monitor French inland surface waters contamination. The survey reveals a chronic state of contamination by estrone (96 % quantification rate) and significant one by 17-β-estradiol and 17-α-ethinylestradiol (15 % quantification rate). Maximum measured concentrations are above predicted no-effect concentrations, indicating potential risk toward the environment.

Optimized method for determination of 16 FDA polycyclic aromatic hydrocarbons (PAHs) in mainstream cigarette smoke by gas chromatography\u2013mass spectrometry

A gas chromatography–mass spectrometry (GC–MS) method was validated for the determination of 16 polycyclic aromatic hydrocarbons (PAHs) from the FDA list of 93 harmful or potentially harmful constituents of mainstream cigarette smoke (MCS). Target analytes were extracted from total particulate matter using accelerated solvent extraction with a toluene/ethanol solvent mixture. Matrix artefacts were removed by two-step solid-phase extraction process. Three different GC–MS systems [GC–MS (single quadrupole), GC–MS/MS (triple quadrupole) and GC–HRMS (high resolution, magnetic sector)] using the same separation conditions were compared for the analysis of MCS of 3R4F Kentucky reference cigarettes generated under ISO and intense smoking regimes. The high mass resolution (m/∆m ≥ 10,000) and associated selectivity of detection by GC–HRMS provided the highest quality data for the target PAHs in MCS. Owing to the HR data acquisition mode enabling measurement of accurate mass, limits of quantification for PAHs were 5 to 15-fold lower for GC–HRMS than for GC–MS/MS and GC–MS. The presented study illustrates that the optimised sample preparation strategy followed by GC–HRMS analysis provides a fit-for-purpose and robust analytical approach allowing measurement of PAHs at (ultra)low concentrations in MCS. Furthermore, the study illustrates the importance and benefits of robust sample preparation and clean-up to compensate for limited selectivity when low-resolution MS is used.

MASS SPECTROMETRY-BASED METHOD TO MEASURE CIRCULATING OXYSTEROLS IN ALZHEIMER’S DISEASE

Hypercholesterolemia is an established risk factor for Alzheimer's disease (AD). Unlike cholesterol, oxysterols; oxygenated derivatives of cholesterols are able to pass the blood brain barrier. Oxysterols are involved in many biological processes including the regulation of cholesterol homeostasis within the brain. However, the modifications to cholesterol are not fully explored due to challenges in detecting these entities at low physiological abundance. The aim of this project is to develop and validate a multiple reaction monitoring (MRM) based liquid chromatography-mass spectrometry (LC-MS/MS) method for the absolute quantification of oxysterols (24OHC, 25OHC, 27OHC, 7βOHC and 7-keto-OHC) in human plasma. A multiple reaction monitoring (MRM) based liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for the absolute quantification of five oxysterols (24OHC, 25OHC, 27OHC, 7βOHC and 7-keto-OHC) in human plasma. Blood from healthy control, age-, sex-matched AD and AD with vascular complications (AD plus) subjects (n=10/group) was collected. Total oxysterols were extracted from plasma (70μl) spiked with internal standards (24OHC-d7, 25OHC-d6, 27OHC-d6, 7βOHC-d7, and 7-keto-OHC-d5) and oxysterols were enriched using two-step solid phase extraction (SPE) using a polymeric SPE column (HLB PRiME, Waters). MRM assay has a linear dynamic range (R2 > 0.94) of 0.02ng/ml-20ng/ml for the five oxysterols tested from. High intra- and inter-day precision (CV < 9%) and high concentration accuracy (< 15% absolute error) were observed from the QC plasma samples. All five oxysterols measured in AD and AD plus patients were not significantly different from healthy control subjects (27±5pg/ml 24OHC, 104±9pg/ml 25OHC, 150±21pg/ml 27OHC, 310±39pg/ml 7βOHC, and 342±42pg/ml 7-keto-OHC). The method described has been validated for five oxysterol measurement in human plasma. These data suggest that patients with Alzheimer's disease have similar levels of oxysterols to healthy individuals.

Determination of Underivatized Glyphosate Residues in Plant-Derived Food with Low Matrix Effect by Solid Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry

A method was developed for the determination of glyphosate residues in plant-derived food using a two-step solid phase extraction (SPE) combined with mixed-mode liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted with water. Then, the extracting solution was pretreated by a C18 cartridge to remove protein and weak-polar interferences and further directly extracted using a strong anion exchange (SAX) cartridge to remove neutral and basic substances. The obtained glyphosate residues from the SPE were separated on a hydrophilic interaction/weak anion-exchange (HILIC/WAX) column and detected by mass spectrometry with negative electrospray ionization (ESI-) in multiple reaction monitoring (MRM) mode. This approach was evaluated by five different kinds of plant-derived food (soybean, corn, carrot, apple, and spicy cabbage) matrices in terms of matrix effect and recovery. Results showed that two-step SPE and mixed-mode chromatography separation provided the method with a very low matrix effect, and the spiked recoveries of glyphosate were satisfied in the range of 83.1 to 100.8 % at three spiked levels. The limit of quantification (LOQ) and detection (LOD) of the method in different matrices were 0.016–0.026 and 0.005–0.008 mg kg−1, respectively. The procedure was validated and showed good accuracy and precision over a large linear range of 0.02–10 mg kg−1.

Analysis of glyphosate and aminomethylphosphonic acid in leaves from Coffea arabica using high performance liquid chromatography with quadrupole mass spectrometry detection

Glyphosate is a commonly applied herbicide in coffee plantations. Because of its non-selective mode of action it can damage the crop exposed through spray drift. Therefore, it is of interest to study glyphosate fate in coffee plants. The aim of this study was to develop an analytical method for accurate and precise quantification of glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) at trace levels in coffee leaves using liquid chromatography with single-quadrupole mass spectrometry detection. The method is based on a two-step solid phase extraction (SPE) with an intermediate derivatization reaction using 9-fluorenylmethylchloroformate (FMOC). An isotope dilution method was used to account for matrix effects and to enhance the confidence in analyte identification. The limit of quantification (LOQ) for glyphosate and AMPA in coffee leaves was 41 and 111μgkg−1 dry weight, respectively. For the method optimization a design of experiments (DOE) approach was used. The sample clean-up procedure can be simplified for the analysis of less challenging matrices, for laboratories having a tandem mass spectrometry detector and for cases in which quantification limits above 0.1mgkg−1 are acceptable, which is often the case for glyphosate. The method is robust, possesses high identification confidence, while being suitable for most commercial and academic laboratories. All leaf samples from five coffee fields analyzed (n=21) contained glyphosate, while AMPA was absent. The simplified clean-up procedure was successfully validated for coffee leaves, rice, black beans and river water.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct detection of 2-monochloropropanediol (2-MCPD) esters in edible oils.

A new analytical method has been developed and validated for the detection and quantification of 2-monochloropropanediol (2-MCPD) esters in edible oils. The target compounds are potentially carcinogenic contaminants formed during the processing of edible oils. As the 2-MCPD esters that occur most frequently in refined edible oils were not commercially available, standards were synthesized with identity and purity (95+%) confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (1)H NMR. Target analytes are separated from edible oil matrices using a two-step solid-phase extraction (SPE) procedure. The extracts are then analyzed using LC-MS/MS with electrospray ionization (ESI). The method has been validated for 11 2-MCPD diesters and 3 2-MCPD monoesters in soybean oil, olive oil, and palm oil using an external calibration curve. The ranges of average recoveries and relative standard deviations (RSD) across the three oil matrices at three spiking concentrations are 79-106% (3-13% RSD) for the 2-MCPD diesters and 72-108% (4-17% RSD) for the 2-MCPD monoesters, with limits of quantitation at or below 30 ng/g for the diesters and 90 ng/g for the monoesters.

Determination of 4-nonylphenols in sediments from a eutrophic marine area

The aim of this work was to develop a simple and efficient analytical method for the determination of 4-nonylphenols (4-NPs) in marine sediments. Nonylphenols are a group of xenobiotics of great environmental concern owing to their endocrine-disrupting properties; they are recognised as priority pollutants by the Water Framework Directive. The analytical procedure involves ultrasonic extraction followed by two-step solid phase extraction clean-up and gas chromatography–mass spectrometry analysis, without a derivatisation step. The method was validated and demonstrated to be suitable for marine sediments rich in organic matter from a eutrophic, contaminated area; 4-NP recovery rates were above 90%. This method was applied to the analysis of 4-NPs in recent sediments from the Gulf of Gdańsk (Southern Baltic Sea). Sediments from this area are moderately contaminated with 4-NPs (0–1 cm layer: 1–42 ng/g d.w.; 1–5 cm layer: 2–61 ng/g d.w.).

Analysis of Organophosphate Esters in Dust, Soil and Sediment Samples Using Gas Chromatography Coupled with Mass Spectrometry

Background contamination is a major problem in the analysis of organophosphate esters (OPEs). In this study, the possible sources of OPEs pollution were screened, and several different ways were applied to minimize the blank contamination. Under the strict quality control conditions, the cleanup efficiency of different solid phase extraction (SPE) was investigated. On the basis of this, a method was developed for the determination of 7 OPEs in dust, soil and sediment samples by gas chromatography coupled with mass spectrometry (GC/MS). The target compounds were extracted by hexane-dichloromethane (1:1, V/V) followed by aminopropyl silica gel SPE column cleanup for dust, and target compounds in soil and sediment were Soxhlet extracted and cleanuped by two-step SPE. The results showed that aminopropyl silica gel SPE column displayed the best purification performance among the three employed columns. Instrumental detection limits of the 7 OPEs ranged from 2.5 to 25.8 μg L−1, and method limits of quantification (MLOQs) in dust and soil sample ranged from 1.4 to 15.7 ng g−1 and 0.3 to 2.9 ng g−1, respectively. The average recoveries of 7 OPEs in different matrices (dust and soil) at two spiked concentration levels ranged from 67.9% to 117.4%. The proposed method was successfully applied to analyze OPEs in different environmental samples collected in Shanghai.

Quantitative high-performance liquid chromatography–tandem mass spectrometry method for the analysis of free desmosines in plasma and urine

A rapid method for the determination of the sum of free desmosine and isodesmosine in human plasma and urine is described. Efficient sample clean-up prior to LC–MS/MS analysis is mandatory for detection of free desmosines in plasma samples. The combination of ultra-filtration and a two-step solid phase extraction minimizes the sample complexity and ion suppression effects. The flow through from the ultra filtration is passed through a C18 resin and then the target analytes are trapped and enriched on a mixed mode solid phase extraction material. The combination of these three orthogonal sample preparation steps allows detection of endogenous free desmosines in plasma from healthy individuals. An analytical column packed with porous graphitic carbon material enables the retention of the polar desmosine analytes, which are measured by electrospray ionization tandem mass spectrometry. Deuterium labeled isodesmosine is added as internal standard and a linear calibration curve was constructed in the range of 0.1–2.0nmol/L for plasma samples and 5–200nmol/L for urine samples. These results demonstrate that the described LC–MS/MS method provides sensitive, repeatable and accurate quantification of free desmosines in plasma and urine samples.

Analysis of Processing Contaminants in Edible Oils. Part 2. Liquid Chromatography–Tandem Mass Spectrometry Method for the Direct Detection of 3-Monochloropropanediol and 2-Monochloropropanediol Diesters

A method was developed and validated for the detection of fatty acid diesters of 2-monochloropropanediol (2-MCPD) and 3-monochloropropanediol (3-MCPD) in edible oils. These analytes are potentially carcinogenic chemical contaminants formed during edible oil processing. After separation from oil matrices using a two-step solid-phase extraction (SPE) procedure, the target compounds are quantitated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). The first chromatographic conditions have been developed that separate intact diesters of 2-MCPD and 3-MCPD, allowing for their individual quantitation. The method has been validated for 28 3-MCPD diesters of lauric, myristic, palmitic, linolenic, linoleic, oleic, and stearic acids in coconut, olive, and palm oils, as well as 3 2-MCPD diesters, using an external calibration curve. The range of average recoveries and relative standard deviations (RSDs) across the three oil matrices at three spiking concentrations are 88-118% (2-16% RSD) with maximum limits of quantitation of 30 ng/g (ppb).

Analysis of Processing Contaminants in Edible Oils. Part 1. Liquid Chromatography–Tandem Mass Spectrometry Method for the Direct Detection of 3-Monochloropropanediol Monoesters and Glycidyl Esters

A new analytical method has been developed and validated for the detection of glycidyl esters (GEs) and 3-monochloropropanediol (3-MCPD) monoesters in edible oils. The target compounds represent two classes of potentially carcinogenic chemical contaminants formed during the processing of edible oils. Target analytes are separated from edible oil matrices using a two-step solid-phase extraction (SPE) procedure. The extracts are then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). Chromatographic conditions that separate sn-1 and sn-2 monoesters of 3-MCPD have been developed for the first time. The method has been validated for GEs, sn-1 3-MCPD monoesters of lauric, myristic, linolenic, linoleic, oleic, and stearic acids, and sn-2 3-MCPD monoesters of oleic and palmitic acids in coconut, olive, and palm oils using an external calibration curve. The range of average recoveries and relative standard deviations (RSDs) across the three oil matrices at three spiking concentrations are 84-115% (3-16% RSD) for the GEs, 95-113% (1-10% RSD) for the sn-1 3-MCPD monoesters, and 76.8-103% (5.1-11.2% RSD) for the sn-2 3-MCPD monoesters, with limits of quantitation at or below 30 ng/g for the GEs, 60 ng/g for sn-1 3-MCPD monoesters, and 180 ng/g for sn-2 3-MCPD monoesters.