Solid-phase Extraction Clean-up Research Articles

Quantitative analysis of oxysterols in zebrafish embryos by HPLC-MS/MS

Zebrafish is an in vivo model used in toxicology to estimate the effects of xenobiotics and their teratogenic consequences. The knowledge of the oxysterols profile in zebrafish, during early embryonic stages, provides important information on the role and biological function of these molecules. This work reports the development and validation of a LC-MS/MS method for the determination of 7 different oxysterols in zebrafish embryos. Sample was treated with a combination of liquid/liquid extraction (LLE) followed by micro solid phase extraction (μSPE) clean-up in order to remove matrix interference and obtain a suitable enrichment factor of the analytes. The method was validated on 2 different embryos growing stages, 3–4 and 24 h post fertilization (hpf), as slight differences in terms of recovery and matrix effect were shown. The validation results provided good accuracy (bias ≤17%; 20% at LOQ) and repeatability (≤15%; ≤19% at LOQ), with low LOQs in the range 22 and 65 pg on 100 embryos sample, without any analyte derivatization, demonstrating the suitability of this analytical method as a useful tool to understand the correlation between oxysterols profile and developmental abnormalities induced by xenobiotic exposure.

Determination of 5-HT3 Receptor Antagonists in Human Urine by Porous Graphitic Carbon (PGC) Solid Phase Extraction (SPE) Coupled with High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS)

The quantitative determination of drugs in urine by high performance liquid chromatography electrospray-ionization mass spectrometry (HPLC-ESI-MS) is challenging due to complex matrix interferences and the relatively low concentrations of analytes in urine. In this study, porous graphitic carbon (PGC) was investigated as solid phase extraction (SPE) sorbent before HPLC-ESI-MS determination of three 5-hydroxytryptamine (3) (5-HT3) receptor antagonists from human urine. The factors affecting extraction of the target antagonists were investigated and optimized systematically by using single-factor tests. The optimized SPE procedure was developed for the simultaneous extraction and sample pretreatment of tropisetron, ondansetron, and granisetron in urine by HPLC-ESI-MS/MS. Using the optimized conditions, the matrix effects were reduced by the PGC-based SPE clean-up procedure, with acceptable matrix effects for tropisetron, ondansetron, and granisetron from 96.53% to 98.42%, 93.19% to 94.36%, and 94.48% to 95.60%, respectively. The reported method significantly removes matrix interferences from the urine samples and exhibited satisfactory recovery and process efficiency for the determination of tropisetron, ondansetron, and granisetron. The established pretreatment method provides a promising strategy for the removal of interferences from hydrophilic basic drugs in biological fluids.

Improving the chromatographic selectivity of β-lactam residue analysis in milk using phenyl-column chemistry prior to detection by tandem mass spectrometry.

Analyte isobaric interferences can limit the development of a comprehensive analytical method for the quantitative liquid chromatography-tandem mass spectrometry profiling of an important cohort of veterinary drugs. In this work, a selective chromatographic separation was developed for the analysis of 32 β-lactam antibiotic residues (12 penicillins, 14 cephalosporins, five carbapenems and faropenem) in milk samples. A range of analytical columns with different stationary phases and mobile phases were evaluated for retention and separation of the β-lactam compounds. Results showed that, among the columns tested, only phenyl-hexyl could adequately separate ampicillin from cephalexin and amoxicillin from cefadroxil, which had shown isobaric interferences on a number of stationary phases. Chromatography was performed using a water/acetonitrile binary gradient with formic acid and ammonium acetate. The β-lactam residues were extracted from the milk samples using a water:acetonitrile solution and purified by C18 dispersive solid-phase extraction (d-SPE) clean-up, followed by concentration under nitrogen and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) determination. Analytes were monitored in positive electrospray ionisation mode (ESI(+)). Possible interfering matrix effects were overcome by using 13 internal standards. The method was fully validated according to 2002/657/EC guidelines, showing satisfactory performance characteristics. Under within-laboratory reproducibility conditions, trueness and precision ranged from 91 to 130% and from 1.4 to 38.6%, respectively. Decision limits (CCα) were in the range 2.1-133μgkg-1. Limits of detection (LODs) and quantitation (LOQs) ranged between 0.0090 and 1.5μgkg-1 and from 0.030 to 5.0μgkg-1, respectively.

Exposure to Mycotoxin-Mixtures via Breast Milk: An Ultra-Sensitive LC-MS/MS Biomonitoring Approach.

Exposure to natural food contaminants during infancy may influence health consequences later in life. Hence, breast milk may serve as a vehicle to transport these contaminants, including mycotoxins, from mothers to their infants. Analytical methods mostly focused on single exposures in the past, thus neglecting co-occurrences and mixture effects. Here, we present a highly sensitive multi-biomarker approach by a sophisticated combination of steps during sample preparation including a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction followed by a solid phase extraction (SPE) cleanup and utilizing stable isotopes for compensating challenging matrix effects. The assay was validated in-house, reaching limits of detection (LOD) for all 34 analytes in the range of 0.1 to 300 ng/L with satisfying extraction efficiencies (75–109%) and stable intermediate precisions (1–18%) for most analytes. Compared to a similar multi-mycotoxin assay for breast milk, LOD values were decreased by a factor of 2–60x enabling the assessment of chronic low-dose exposures. The new method was applied to a small set of Nigerian breast milk samples (n = 3) to compare results with already published data. Concentration levels of samples that were found to be contaminated before could be confirmed. In addition, other mycotoxins were determined in all three samples, for example the newly investigated alternariol monomethyl ether (AME) was found for the first time in this biological fluid at concentrations up to 25 ng/L. Moreover, in a pooled Austrian sample obtained from a milk bank, trace amounts of multiple mycotoxins including AME (1.9 ng/L), beauvericin (5.4 ng/L), enniatin B (4.7 ng/L), enniatin B1 (<LOQ), ochratoxin A (<LOQ) and the estrogenic zearalenone (<LOQ) confirmed co-occurrence and exposure even in a country with high food safety standards. In conclusion, the method facilitates the determination of mycotoxins at ultra-trace levels in breast milk, enabling the generation of occurrence data necessary for comprehensive co-exposure assessment.

Quantifications of saxitoxin concentrations in bivalves by high performance liquid chromatography-tandem mass spectrometry with the purification of immunoaffinity column

In this study, saxitoxin (STX) immunoaffinity column (IAC) solid phase extraction (SPE) technology was used to extract and purify STX in bivalve aquatic products. By optimizing the conditions of sample pretreatment, the method of detecting STX in bivalve aquatic products had been established by high performance liquid chromatograph-tandem mass spectrometry (LC-MS/MS) based on the cleanup of SPE with immunoaffinity interaction mechanism. The phosphate buffer solution (PBS) was used to extract STX in bivalves. The sample was separated on a TSK-GEL Amide column (2.0 mm × 250 mm, 5 µm) with water contained 2 mM ammonium formate-50 mM formic acid and 95% acetonitrile contained 2 mM ammonium formate-50 mM formic acid as mobile phase by gradient elution. STX had a good linearity in the range of 2.468 μg/kg to 246.8 μg/kg with the correlation coefficient of r greater than 0.999. The detection of limit (0.1 μg/kg) was more sensitive, two orders of magnitude better than previous report (20 μg/kg) in bivalve aquatic products. The recovery ranged from 79.3% to 102.9%. The method has a good selectivity with eliminating matrix effect thoroughly, no need for matrix matching, thus it can satisfy the requirements of trace STX detection in bivalve aquatic products.

Simultaneous determination of nine perfluoroalkyl carboxylic acids by a series of amide acetals derivatization and gas chromatography tandem mass spectrometry

A novel and simple derivatization method using a series of amide acetals as derivatization reagents, along with gas chromatography tandem mass spectrometry (GC–MS/MS) analysis, were developed and validated for simultaneous determination of 9 perfluoroalkyl carboxylic acids (PFCAs) in this study. The structures and fragmentation pathway of PFCAs derivatives were deduced and verified. The derivatization method developed in this study improved the sensitivity of the detection of PFCAs by GC. The applicability of 6 amide acetals for the derivatization of PFCAs was demonstrated. Derivatization conditions for 9 PFCAs were optimized with addition of 20 μL of derivatization reagent and reaction at 35 °C for 30 min. 9 PFCAs derivatives were confirmed to be stable over 15 days. The instrument detection limits (IDLs) were lower than 0.01 pg/μL. The intra and inter-day precisions were below 4.06% and 5.48%, respectively. To demonstrate the utility of the derivatization method, the level of PFCAs in the marine products were detected. The alkaline digestion followed by solid-phase extraction (SPE) cleanup method was used for pretreatment. The method detection limits (MDLs) ranged from 0.04 to 0.10 ng/g, and the spiked recoveries ranged between 54.72% and 107.29%, with relative standard deviation (RSD) of 1.53%-11.89%. Five PFCAs were detected in the range of 0.66 to 499.03 ng/g dry weight.

DLLME-μSPE extraction coupled to HPLC-ESI-MS/MS for the determination of F2α-IsoPs in human urine

Oxidative stress is a pathological condition characterized by an imbalance between body’s antioxidant defenses and oxidizing agents, resulting in damage of endogenous molecules. These products can be used as markers of oxidative conditions; in particular, isoprostanes (IsoPs) come from the reaction of arachidonic acid with reactive oxygen species (ROS) and are currently defined as gold markers of oxidative stress in urine. Our main goal was the development of a reliable analytical method for the determination and quantification of the IsoPs in human urine by dispersive Liquid–Liquid Micro Extraction (dLLME) coupled with micro Solid Phase Extraction (μSPE) clean-up and HPLC-MS/MS analysis. The selected compounds are present in very small concentration in urine, furthermore, due to relevant matrix effect, they are challenging for ESI-MS/MS analysis. This approach provided selectivity and sensitivity for 8-isoprotaglandine F2α (8-iso-PGF2α), the “gold” OS marker, together with the main isomers. dLLME extraction allowed a significant enrichment factor and μSPE clean-up provided the removal of ion-suppressing compounds from the sample resulting in low matrix effect. The chromatographic separation was also challenging as the target compounds possess very similar chemical characteristics, so experimental conditions were carefully tuned. The reported method represents a useful tool for the detection of IsoPs in urine taking advantage of the combination of dLLME extraction and μSPE clean-up; overall recoveries were above 50 % and matrix effects were ≤15 %, with LOQs ranging between 0.020 and 0.060 ng mL−1. The procedure is easy to use and rapid allowing the removal of interfering compounds and matrix effect maintaining a highly sensitive determination.

Determination of polycyclic aromatic hydrocarbons in traditionally smoked meat products and charcoal grilled meat in Cyprus

The study highlights the investigation of the presence of polycyclic aromatic hydrocarbons (PAHs) in traditionally smoked meat products and charcoal grilled meat. Specifically, the levels of benzo(a)anthracene (BaA), chrysene (Chr), benzo(b)fluoranthene (BbF) and benzo(a)pyrene (BaP) were determined for 262 samples of smoked sausage, bacon, pork (lountza) and ham (chiromeri), grilled pork and poultry produced in Cyprus. The method is based on a saponification and liquid extraction step, followed by solid phase extraction (SPE) cleanup of the extract and finally, the determination of PAHs using high performance liquid chromatography with fluorescence detector. Most of the samples analysed (96%) were contaminated with at least one PAH. In these contaminated samples, 12% of the smoked products and 15% of the grilled meat samples exceeded the maximum levels of the EU legislation. The highest PAH concentrations were found in samples with higher fat content and longer smoking or cooking time.

Impact of Raw Materials and Production Processes on Furan and Acrylamide Contents in Dark Chocolate.

This study was aimed to evaluate the level of furan and acrylamide contamination in cocoa and noncocoa raw materials, in masses from processing stages, and in chocolates originating from three factories. Acrylamide was determined by the gas chromatography-mass spectrometry (GC-MS) method using the QuEChERS procedure with dispersive solid-phase extraction clean-up and isotopic standard (2,3,3-d3-acrylamide). Furan was analyzed by the headspace solid-phase microextraction/GC-MS technique with the d4-furan marker. Both analytical methods were validated in terms of accuracy, precision, and linearity as well as the limit of detection (LOD) and limit of quantification (LOQ). Among all raw materials, the most abundant in acrylamide were cocoa masses and powders (83.0-127.5 ng g-1). Roasting of cocoa beans increased the content of acrylamide 2-3-fold. The obtained results indicate that acrylamide might be formed during wet conching. Only in cocoa powders and lecithin, it was possible to quantify furan (3.7-10.2 and 16.3 ng g-1, respectively). Roasting of cocoa beans increased the content of furan from <LOD to 25.1-34.8 ng g-1. Because of the high volatility of furan and specific manufacturing processes, favorable conditions were created for furan evaporation. Manufactured chocolates were products with a low level of acrylamide (61.8-108.0 ng g-1) and a very low level of furan (<1.5 ng g-1).

A newly modified QuEChERS method for the analysis of organochlorine and organophosphate pesticide residues in fruits and vegetables.

A newly modified QuEChERS method was developed, validated, and tested in-house for the multiclass, multiresidue determination of organophosphate (OPP) and organochlorine pesticide (OCP) residues in fruits and vegetables. The method incorporated a solvent extraction with acetonitrile followed by partitioning with magnesium sulphate (MgSO4) and sodium chloride (NaCl). The final step involved a dispersive solid phase extraction (d-SPE) clean-up prior to gas chromatography with electron capture detection (GC-ECD) analysis. Apples and lettuce were the selected matrices for fruits and vegetables, respectively in the method development stages. Various combinations of sorbents were tested in the clean-up step. Florisil and MgSO4 (FM) d-SPE proved to be the best combination for the clean-up step for both OCPs and OPPs in both the fruit and vegetable matrices. Recovery values fell within the acceptable range of 70 to 120% (RSD ≤20%). The new method is unique as it employs the standard addition calibration technique for the quantification of OCP and OPP residues. Limit of detection, limit of quantification, linearity, recovery analysis, and repeatability were validation parameters used to evaluate the accuracy, precision, and ruggedness of the method. The newly modified QuEChERS method provides a cheaper alternative for the analysis of pesticide residues in fruits and vegetables as samples can be analysed at a fraction of the cost of the original QuEChERS method. The efficacy of the method was tested on several fruits and vegetables from the Central Division of Trinidad and Tobago. OCP and OPP residues were found in 61% of the samples, most of which were above the maximum residue limits.

Screening for More than 1,000 Pesticides and Environmental Contaminants in Cannabis by GC/Q-TOF

A method has been developed to screen cannabis extracts for more than 1,000 pesticides and environmental pollutants using a gas chromatograph coupled to a high-resolution accurate mass quadrupole time-of-flight mass spectrometer (GC/Q-TOF). An extraction procedure was developed using acetonitrile with solid phase extraction cleanup. Before analysis, extracts were diluted 125:1 with solvent. Two data mining approaches were used together with a retention-time-locked Personal Compound Database and Library (PCDL) containing high-resolution accurate mass spectra for pesticides and other environmental pollutants. (1) A Find-by-Fragments (FbF) software tool extracts several characteristic exact mass ions within a small retention time window where the compound elutes. For each compound in the PCDL, the software evaluates the peak shape and retention time of each ion as well as the monoisotopic exact mass, ion ratios, and other factors to decide if the compound is present or not. (2) A separate approach used Unknowns Analysis (UA) software with a peak-finding algorithm called SureMass to deconvolute peaks in the chromatogram. The accurate mass spectra were searched against the PCDL using spectral matching and retention time as filters. A subset PCDL was generated containing only pesticides that are most likely to be found on foods in the US. With about 250 compounds in the smaller PCDL, there were fewer hits for non-pesticides, and data review was much faster. Organically grown cannabis was used for method development. Twenty-one confiscated cannabis samples were analyzed and ten were found to have no detectable pesticides. The remaining 11 samples had at least one pesticide and one sample had seven detectable residues. Quantitative analysis was run on the confiscated samples for a subset of the pesticides found by screening. Two cannabis samples had residues of carbaryl and malathion that were estimated to be about 10 times greater than the highest US Environmental Protection Agency tolerance set for food and about 4,000 times greater than the Canadian maximum residue limits for dried cannabis flower.

Analysis of artificially oxidized cardiolipins and monolyso-cardiolipins via liquid chromatography/high-resolution mass spectrometry and Kendrick mass defect plots after hydrophilic interaction liquid chromatography based sample preparation.

Cardiolipins (CL) are a special lipid class which plays a main role in energy metabolism in mitochondria and is involved in apoptosis. In contrast to other glycerophospholipids, they contain four fatty acyl residues which results in a high structural diversity. Oxidation, for example by reactive oxygen species, or lyso forms such as monolyso-CL (MLCL), increases this diversity. Mass spectrometric analysis and computational identification of CL, MLCL and their oxidation products is therefore a challenging task. In order to distinguish CL, MLCL and their oxidation products, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed. A hydrophilic interaction liquid chromatography (HILIC)-based solid-phase extraction (SPE) clean-up approach was developed for CL enrichment. Graphical analysis of CL, MLCL and their oxidation products was carried out by a three-dimensional Kendrick mass defect (3D-KMD) plot module, as well as a refined lipid search module of the open-source metabolomics data mining software MZmine 2. The HILIC-based SPE clean-up enabled complete separation of polar and nonpolar lipid classes. A yeast (Saccharomyces cerevisiae) lipid extract, which was artificially oxidized by means of the Fenton reaction, was analyzed by the developed LC/MS/MS method. CL species with differences in chain length and degree of unsaturation have been separated by high-performance liquid chromatography (HPLC). In total 66 CL, MLCL and oxidized species have been identified utilizing 3D-KMD plots in combination with database matching using MZmine 2. For further characterization of annotated species, MS/MS experiments have been utilized. 3D-KMD plots capturing chromatographic and high-resolution mass spectrometry data have been successfully used for graphical identification of CL, MLCL as well as their oxidized species. Therefore, we chose multiple KMD bases such as hydrogen and oxygen to visualize the degree of unsaturation and oxidation capturing chromatographic data by means of a color-coded paint scale as the third dimension. In combination with database matching, the analysis of low concentrated lipid species in complex samples has been significantly improved.

Simultaneous determination of multiclass antibiotics in sewage sludge based on QuEChERS extraction and liquid chromatography-tandem mass spectrometry

A QuEChERS based method with dispersive solid-phase extraction (d-SPE) clean-up followed by LC-MS-MS analysis was developed for simultaneous determination of seventeen multiclass antibiotics in sewage sludge and applied to samples from Nigeria.

Application of Automated Mini-Solid-Phase Extraction Cleanup for the Analysis of Pesticides in Complex Spice Matrixes by GC-MS/MS.

Pesticide residues are routinely tested in spices for trade compliance. This results in a huge sample load for food testing laboratories and demands automation in sample preparation. Although there exists a method for the analysis of pesticides in fruits using an automated sample cleanup by mini-solid-phase extraction (mini-SPE) technique, no study is available to date on spices. This study aims to develop an automated sample cleanup method using mini-SPE technique in a range of spices, including chili powder, turmeric, black pepper, cumin, coriander, and cardamom. This automated sample preparation workflow involved an X-Y-Z instrument autosampler, and a set of mini-SPE cartridges comprising cleanup sorbents. Spice samples were extracted by acetonitrile, and the extract was put into an autosampler vial for automated mini-SPE cleanup before analysis by GC tandem MS. For an efficient cleanup, three different sorbent compositions were compared along with various automated workflows. For the relatively simple matrixes (e.g., coriander, cumin, and cardamom), the LOQ for the target pesticides was 10 ng/g with acceptable recovery, and precision. The method provided an LOQ of 10 ng/g for around 77% of the compounds in the relatively complex matrixes (e.g., turmeric, chili powder, and black pepper). The remainder of the compounds had satisfactory recoveries at 20 ng/g and higher levels. Given its time effectiveness and efficient analytical performance, this method can be adopted in commercial food testing laboratories for time-bound analysis of a large volume of samples. The study describes effectiveness of the automated mini-SPE cleanup in multiresidue analysis of pesticides in a range of spice matrixes. The method facilitates high-throughput residue analysis in compliance with the regulatory requirements of sensitivity and method performance.

Single-Laboratory Validation of AOAC Official Method 2011.10 for Vitamin B12 in 'Indian' Infant and Pediatric Formulas and Adult Nutritionals.

Ensuring the quality of infant and pediatric formulas and adult nutritionals is of utmost importance for the health and safety of rapidly urbanizing Indian population. B12 is an important water-soluble vitamin, which is fortified externally in such nutritional formulations. The Bureau of Indian Standards (BIS) has a recommended microbiological assay-based method for determination of vitamin B12 that is not precise and accurate enough to meet the label claim requirements of infant, adult, and/or pediatric nutritionals. The AOAC Official Method 2011.10 was originally developed under the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) for the determination of vitamin B12 in infant and pediatric formulas and adult nutritionals. However, those SPIFAN matrixes did not contain malt and other indigenous cereal and legume flour (with or without cocoa powder), which are commonly found in Indian formulations. Thus, there is a need to replace this method with a more precise and accurate method. This study was undertaken to validate the AOAC Official Method 2011.10 on vitamin B12 in 'Indian' infant and pediatric formulas and adult nutritionals. The single-laboratory validation (SLV) of AOAC Method 2011.10 was carried out as per the AOAC Guidelines in six Indian pediatric and adult nutritional formulas to verify its fitness for purpose. Cobalamin in the sample was converted to cyanocobalamin on treatment with potassium cyanide. The sample was then subjected to clean up through a C18 cartridge. Vitamin B12 in the eluted extract was separated from other components using size-exclusion column chromatography followed by a C18 column. The HPLC analysis was carried out at 550 nm. Diastase treatment and C18 solid-phase extraction cleanup satisfactorily removed the matrix interference. The relative standard deviation of the determined values in 30 samples each from 6 selected Indian products and NIST SRM 1849a was <20%. The average recoveries for the spiked recovery samples ranged from 91.75 to 101.14%. Method 2011.10 met the standard method performance requirements set forth by the AOAC SPIFAN. Therefore, we recommend the Method 2011.10 for adoption as the BIS official method for the analysis of vitamin B12 in 'Indian' infant and pediatric formulas and adult nutritionals. This was the first SLV project that the AOAC India section undertook to extend the scope of the AOAC Method 2011.10 for vitamin B12 analysis by validating it in 'Indian' infant and pediatric formulas and adult nutritionals.

Development of an UPLC–MS/MS method for the analysis of 16 synthetic opioids in segmented hair, and evaluation of the polydrug history in fentanyl analogue users

Seizures of synthetic opioids have increased since 2012, with a 45 % increase in synthetic opioid related deaths between 2016 and 2017 in US. Recently, concerns have arisen around these substances and their illicit use also in several European countries.Our aim was to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of 16 synthetic opioids in segmented hair, including fentanyl, norfentanyl, acetylfentanyl, U-47700, AH-7921, acrylfentanyl, crotonylfentanyl, butyrylfentanyl, methoxacetylfentanyl, U-49900, valeryfentanyl, 4-fluoro-iso-butyrylfentanyl, ocfentanyl, furanylfentanyl, tetrahydrofuranylfentanyl, and alfetanyl.Sample preparation involved washing the hair in dichloromethane, water and methanol, and extraction in methanol, followed by solid phase extraction clean-up. This method was validated for linearity, limit of quantification (LLOQ), precision and bias, selectivity, stability, matrix effects, extraction efficiency of the clean up procedure, and carryover. LLOQs ranged from 0.15−1pg/mg, and the calibration ranged from the LLOQ up to 500pg/mg. Intra and inter-day precision were evaluated at low and high concentrations, with spiked QCs, during 8 days and the results were satisfactory with RSD<15 % for all the compounds except for norfentanyl (22 %) and alfentanyl (19 %). Two external certified QCs containing fentanyl at 11 and 105pg/mg were also analysed within each batch and the RSD and bias were lower than 16 % and 10 %, respectively.Matrix effects compensated by internal standard fentanyl-d5 (MEIS), were between 77–115 % (RSD<10 %) and extraction efficiency of the clean-up procedure was between 66–93 % (RSD<21 %). Processed sample stability and carryover were acceptable for all of the compounds.The method was applied to 17 authentic hair samples (body or head hair) from US fentanyl analogue users. When head hair was available, the hair strands were analysed in 1cm/segment. Concentrations ranges were as follows: fentanyl (n=16) 2->ULOQ (500) pg/mg, norfentanyl (n=14) 1−38pg/mg, acetylfentanyl (n=7) 0.6->ULOQ (250) pg/mg, furanylfentanyl (n=5) 2−123pg/mg, tetrahydrofuranylfentanyl (n=1) 0.5−63pg/mg and valerylfentanyl (n=1) 2.1->ULOQ (50) pg/mg, along the hair strands. To our knowledge, this is the first time where concentrations of tetrahydrofuranylfentanyl, and valerylfentanyl in hair are reported. The same samples were also analysed for the determination of other drugs of abuse using our routine method (also in 1cm/segment for head hair when available). The results demonstrated poly-drug use in these fentanyl-analogue users population (mean drugs: n=5): amphetamine and/or methamphetamine (n=10), buprenorphine (n=5), cocaine (n=8), methadone (n=8), 6-MAM (n=17), meperidine (n=1), oxycodone (n=11), tramadol (n=3). Evaluation of the concentrations of these drugs, together with the fentanyl analogues is discussed in the present paper.Two authentic samples from two Belgian post-mortem cases, were also analysed showing fentanyl use and in one case polydrug use.The results demonstrated multi-analyte quantitative methods, including fentanyl analogues, are becoming useful in forensic laboratories involved in hair analysis, and in particular when polydrug use is suspected.

Development and validation of a SPE-UHPLC-fluorescence method for the analysis of ochratoxin-a in certain Turkish wines

A sensitive Ultra-High Performance Liquid Chromatography (UHPLC)-fluorescence method was developed and validated for the determination of ochratoxin-A (OTA) in Turkish wine samples. Naphthalene was used as an internal standard in this study. OTA was separated on a C18 (3.0 mm × 100 mm × 1.8 µm) column and analyses were run under isocratic conditions, with a mobile phase consisting of water/acetonitrile/acetic acid (50:50:1, v/v/v). The flow rate and injection volume were 0.5 ml min−1 and 10 μl, respectively. The excitation and emission wavelengths were 330 nm and 460 nm for OTA, respectively, and 220 nm and 325 nm for internal standard, respectively. A solid-phase extraction (SPE) clean-up procedure on a C18 cartridge was used prior to the analysis of the wine samples by UHPLC. The developed method was validated with respect to linearity, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ), stability and robustness. The method presented good RSD (&lt; 4 %) and recovery (102.6–105.2 %) values. The LOD and LOQ values were 0.01 ng ml–1 and 0.05 ng ml–1, respectively. All other parameters were acceptable. OTA amounts were found in the range of 2.72‒7.40 µg kg‒1 in the Turkish wine samples.

Modified QuEChERS method for phenolic compounds determination in mustard greens (Brassica juncea) using UHPLC-MS/MS

A modified QuEChERS method (Quick, Easy, Cheap, Effective, Rugged, and Safe) for the determination of fifteen phenolic compounds in mustard greens (Brassica juncea) using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) analysis was developed. The QuEChERS partitioning step and dispersive solid phase extraction (d-SPE) clean-up sorbents were investigated, aimed at phenolic compound extraction and pigment removal, respectively. QuEChERS acetate version combined with 25 mg of diatomaceous earth (DE) and 5.0 mg of graphitized carbon black (GCB) provided the best conditions for sample preparation of the target compounds. Under the optimized conditions, all phenolic compounds showed good linearity (r ≥ 0.99) over the concentration range of 0.1 to 8000 μg kg−1, and the quantification limits were in the range of 0.06–230 μg kg−1. The spectrophotometric analysis showed that the clean-up step promoted a significant removal of chlorophyll, which is the major pigment present in the sample. Furthermore, antioxidant activity analysis was also carried out after the clean-up step and, together with chromatographic data, showed that no significant retention of the phenolic compounds occurs in the clean-up step. Two mustard greens varieties – Southern Giant Curled (SGC) and Florida Broadleaf (FB) - were analyzed with the proposed method. Seven phenolic compounds (4-hydroxybenzoic, p-coumaric, ferulic and sinapic acids, naringenin, apigenin and kaempferol) were found in both varieties, the greatest abundance being for sinapic acid (1261.5 ± 23 μg kg−1 in SGC and 1235.5 ± 26 μg kg−1 in FB) and ferulic acid (2861 ± 24 μg kg−1 in SGC and 3204.5 ± 45 μg kg−1 in FB).

Rapid extraction method followed by a d-SPE clean-up step for determination of phenolic composition and antioxidant and antiproliferative activities from berry fruits

Despite the numerous studies that have shown a wide range of biological activities to berry fruits, scientific data focusing on modern, rapid and simple extraction methods followed by a clean-up step is still lacking. Therefore, the present work was aimed at investigating the use of a fast one-step solid–liquid extraction procedure followed by a dispersive solid-phase extraction (d-SPE) clean-up step to evaluate the phenolic composition, antioxidant and antiproliferative activities from three of the principal berries found in Brazil, pomegranate (Punica granatum L.), blackberry (Rubus ulmifolius Schott.), and strawberry (Fragaria × ananassa Duch.). Under the optimized extraction conditions, sixteen phenolic compounds were determined by UHPLC-MS/MS analysis and all berry extracts showed antioxidant activity and antiproliferative effects on cervical (HeLa) and colon (HT-29) cancer cells. Overall, these results highlight the importance of the clean-up step for more reliable data in studies of health-promoting proprieties from berry fruits.

QuEChERS-based gas chromatography-electron capture/flame photometric detection method for multi-pesticide residues analysis in Andrographis paniculata: a popular Indian medicinal herb

ABSTRACT Andrographis paniculata (kalmegh) is a medicinal plant consumed at ton scale worldwide. This herb has a high demand both in traditional ayurvedic formulations and in the pharmaceutical industry. Owing to the continuous global demand for pharmaceutical applications, quality of cultivated kalmegh is very important and the presence of pesticide residues may pose serious restrictions by various national/international regulatory bodies for consumer safety. The European pharmacopoeia, require validated methods to determine the pesticide residues in each specific medicinal plant. In order to achieve such requirements, a modified quick, easy, cheap, efficient, rugged and safe (QuEChERS) method coupled with gas chromatography with electron capture detector or flame photometric detection (GC–ECD/FPD) was developed for the determination of the multiclass pesticide residues of 12 organochlorines (OCs), 8 organophosphates (OPs), 6 synthetic pyrethroids (SPs) insecticides, 5 herbicides, and 3 fungicides in kalmegh. Initially, pesticides were extracted with acetonitrile and then, the extracts were purified by dispersive solid-phase extraction clean-up. The developed method showed good linearity for all pesticides with high values of correlation coefficients (0.973–0.997) coupled with low limits of detection (LOD) and quantification (LOQ) values ranging from 0.001–0.01 µg/mL and 0.01–0.075 µg/g, respectively. Matrix effects were also investigated and matrix-matched calibration curves were used for all studies. Mean recoveries (60.8–111.8%, with RSD 8.1%) were within the international acceptable limits (70–120%) along with uncertainty analysis ranging from 7.69 to 21.15%. The validated established method was effectively employed for determining the pesticide residues in real field samples and can be extended for commercial and regulatory residue analysis.