Analyte Protectants Research Articles

Analyte protectant approach to protect amide-based synthetic cannabinoids from degradation and esterification during GC–MS analysis

The forensic analysis of amide-based synthetic cannabinoids (SCs) in seized materials is routinely performed using gas chromatography-mass spectrometry (GC–MS); however, a major challenge associated with GC–MS is the thermolytic degradation of substances with sensitive functional groups. Herein, we report the comprehensive thermal degradation and ester transformation of amide-based SCs, such as AB-FUBINACA, AB-CHMINACA, and MAB-CHMINACA, during GC–MS analysis and their treatment with analyte protectants (APs). These SCs were found to undergo thermolytic degradation during GC–MS in the presence of non-alcohol solvents. Using methanol as an injection solvent resulted in the conversion of the amide group to an ester group, producing other SCs such as AMB-FUBINACA, MA-CHMINACA, and MDMB-CHMINACA. Degradant and ester product formation has been interpreted as the adsorption of target SCs on glass wool via hydrogen bonding interactions between the active silanol and amide groups of the SCs, followed by an addition and/or elimination process. The factors found to influence the thermal degradation and/or esterification of the amide functional group include residence time, activity of glass wool, and injection volume. This report presents the fragmentation patterns of all compounds that were produced by degradation and esterification. Using 0.5 % sorbitol (AP) in MeOH as an injection solvent resulted in complete protection and improvement of the chromatographic shape of the compounds. This method has been successfully confirmed in terms of sensitivity, linearity, accuracy, and precision for standard solutions and tablet extraction using 0.5 % sorbitol in MeOH. Using AP increased the sensitivity by ten times or more compared to the use of only MeOH. The limit of detection for all analytes was determined as 25 ng/mL, and the calibration curves were linear over the concentration range of 50–2000 ng/mL. The values of accuracy error were below 11 %, and precision was less than 13 %. The effects of phytochemicals of herbal products, tablet ingredients, and biological matrices on the degradation and/or esterification and APs performance have also been evaluated in this work.

Development of an enhanced analytical method utilizing pepper matrix as an analyte protectant for sensitive GC‒MS/MS detection of dimethipin in animal-based food products.

Herein, an analytical method using gas chromatography-tandem mass spectrometry (GC‒MS/MS) was devised to detect the presence of the troublesome pesticide dimethipin in various animal-based food products, including chicken, pork, beef, eggs, and milk. The injection port was primed with a matrix derived from pepper leaves that acts as an analyte protectant (AP) to safeguard the target compound from thermal degradation during gas chromatography. The presence of AP resulted in a remarkable limit of quantification of 0.005 mg/kg for dimethipin in five matrices. Three different versions (original, EN, and AOAC) of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method were compared for dimethipin extraction, with a double-layer solid-phase extraction (SPE) cartridge utilized for matrix purification. A seven-point external calibration curve was established for dimethipin in the five matrices, demonstrating excellent linearity with determination coefficients (R2) ≥ 0.998. The developed quantitative method was validated by fortifying each matrix with three different concentrations of standard dimethipin, and the average recovery fell within the acceptable range outlined in the CODEX guidelines (ranging from 88.8% to 110.0%), with a relative standard deviation (RSD) of ≤ 11.97%. This method effectively addresses the challenge of analyzing dimethipin and can therefore be used as a routine monitoring tool for dimethipin across various matrices.

Accuracy improvement of determination of seven minor tobacco alkaloids in mainstream cigarette smoke using analyte protectants by gas chromatography-mass spectrometry

Tobacco alkaloids are important precursors of carcinogenic tobacco-specific nitrosamines. Therefore, accurate quantification of tobacco alkaloids is highly important. This study investigates the compensation effects of novel analyte protectants (APs) for matrix effects (MEs) to determine seven minor tobacco alkaloids (nornicotine, myosmine, anabasine, anatabine, nicotyrine, 2,3′-bipyridine, and cotinine) in mainstream cigarette smoke with high accuracy and robustness. By comparing the heights and shapes of the chromatographic peaks before and after the addition of APs to standard solutions prepared in dichloromethane and cigarette smoke solutions, the compensation effects of 12 APs and their combinations on the MEs of seven minor tobacco alkaloids were evaluated, and the best combination of 2-pyridylethylamine (2 mg/mL)+1,2-decanediol (1 mg/mL) was identified. This AP combination could effectively improve the shapes and increase the heights (by 7–371%) of chromatographic peaks for standard solutions prepared in dichloromethane and cigarette smoke solutions. Before the addition of this AP combination, the slope ratios of the calibration curves for two types of standard solutions of the seven target chemicals were 71.4–159.8%, while they were 87.4–105.6% after the addition, indicating that this AP combination reduced the matrix difference between pure solvent and cigarette smoke solution. After adding the AP combination, the standard curves of solutions prepared in dichloromethane showed good linearity (r2 ≥ 0.999), the spiked recoveries were between 80.9% and 119.6%, and the inter- and intraday precisions were between 1.5–9.5% and 3.1–8.5%, respectively. Three commercial cigarette samples and one mixed standard solution were also tested under four different instrument working conditions to verify the long-term accuracy and ruggedness of the approach in routine real-world analysis of the method. The results showed that the RSD values were higher (3.5–25.4%) without the AP combination than that (<6.7%) with the AP combination. Because of its high accuracy, precision, and robustness, this method has good practical prospects.

Critical review and re-assessment of analyte protectants in gas chromatography

Despite nearly 80 years of advancements in gas chromatography (GC), indirect chemical matrix effects (MEs), known as the matrix-induced response enhancement effect, still occur to cause a high bias in the GC analysis of susceptible analytes, unless precautions are taken. Matrix-matched calibration is one common option used in GC to compensate for the MEs, but this approach is usually inconvenient, imprecise, and inefficient. Other options, such as the method of standard additions, surface deactivation techniques, chemical derivatizations, priming the GC, and/or use of internal standards, also have flaws in practice. When methods are accommodating, the use of analyte protectants (APs) can provide the best practical solution to not only overcome MEs, but also to maximize analyte signal by increasing chromatographic and detection efficiencies for the analytes. APs address the source of MEs in every injection by filling active sites in the GC inlet, column, and detector, particularly in GC–MS, rather than the analytes that would otherwise undergo degradation, peak tailing, and/or diminished response due to interactions with the active sites. The addition of an adequate amount of APs (e.g. sugar derivatives) to all calibration standards and final extracts alike often leads to lower detection limits, better accuracy, narrower peaks, and greater robustness than the other options to compensate for MEs in GC. This article consists of a critical review of the scientific literature, proposal of mechanisms and theory, and re-evaluation studies involving APs for the first time in GC-orbitrap and GC–MS/MS with a high-efficiency ion source design. The findings showed that 1 µg each of co-injected shikimic acid and sorbitol in the former case, and 1 µg shikimic acid alone in the latter case, led to high quality results in multi-residue analysis of pesticides and environmental contaminants.

Combination of analyte protectants and sandwich injection to compensate for matrix effect of pesticides residue in GC–MS/MS

Matrix effect (ME) in gas chromatography (GC) is usually due to undesired interactions with active sites in the inlet and column which can lead to analyte losses and/or peak tailing. Analyte protectants (APs) are compounds added individually or in combination to interact with the active sites and minimize errors related to ME. However, the current Aps mixtures/introducing approach cannot sufficiently compensate for ME in all cases. In this study, a comparison (based on sensitivity and peak shape) between thirteen different Aps (2,3-Butanediol, 3-O-ethylglycerol, d-Fructose, d-Gluconic acid γ-lactone, d-Glucose, d-Ribonic acid γ-lactone, d-Ribose, D-Sorbitol, l-Gulonic acid γ-lactone, Menthol, Polyethylene glycol, Triglycerol and Vanillinin) four different solvents (n-hexane, acetone, acetonitrile and ethyl acetate) for 224 pesticides is presented. The comparison was done between solvent based calibration and extracts of one selected difficult matrices (strawberry), prepared according to the citrate-buffered QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure. Each Ap had different behavior for each solvent. In general, acetonitrile showed the most susceptibility to polar Aps, followed by acetone, then ethyl acetate and then n-hexane. However, in the absence of Aps, peak shapes were best for n-hexane, then ethyl acetate, then acetone and then acetonitrile. Based on their results, a mixture of seven Aps was chosen. To ease their introduction, the sandwich injection approach (SIA) was used. Several factors were studied such as the calibration solvent, Aps solvent and Aps concentration. For acetonitrile calibration, the ME of 213/224 (95%) pesticides were ≤ 20% in one Aps mixture compared to 36 pesticides (16%) in non-protected mixtures. For n-hexane:acetone (9:1) calibration, the ME of 221/224 (99%) pesticides were ≤ 20% in one Aps mixture compared to 102 pesticides (45%) in non-protected mixtures.

A comprehensive analysis of 201 pesticides for different herbal species-ready application using gas chromatography–tandem mass spectrometry coupled with QuEChERs

Developing an analysis of multi-pesticide residues for different herbal species-ready applications is a challenge. In the present work, a comprehensive analysis was proposed for rapid detection of 201 pesticides in various medicinal herbs. Samples were extracted and cleaned up with a high throughput pretreatment approach (modified QuEChERS), and then detected by gas chromatograph coupled to an electron impact ionization triple quadrupole mass spectrometer (GC-EI-MS/MS). The clean-up procedure has been optimized using four types of representative medicinal herbs with different primary or secondary metabolites. Moreover, a mixture of analyte protectants (APs) was to improve the peak shape and intensity of some compounds. The performance of the method was validated according to the European Union SANTE/11813/2017 regulatory guidelines. The limit of quantification (LOQ) was determined to be ≤10 ng mL−1, and the recovery was between 70.0%–120.0%, with ≤20% RSD for the majority of pesticides. Sixty samples belonging to different species of medicinal herbs (such radix, flos, cortex, fructus, and seeds) were analyzed to evaluate the applicability of the optimized method. High frequency of chlorpyrifos was found in Citri reticulatae pericarpium, Crataegi fructus and Cuscutae semen samples.

Three approaches to minimize matrix effects in residue analysis of multiclass pesticides in dried complex matrices using gas chromatography tandem mass spectrometry

This paper discusses one of the major concerns in pesticide residue analysis: the matrix effect related to gas chromatography (GC), which can adversely affect quantification. In this study, a comparison of approaches for dealing with the matrix effect was investigated for 236 pesticides in complex matrices, including dried herbs (Centaurea cyanus L., Matricaria chamomilla L., Thymus vulgaris L.) and dried fruit (currants, chokeberry), using a modified QuEChERS method and GC–MS/MS analysis. Three approaches were evaluated: (i) using matrix-matched calibration, (ii) adding a mixture of analyte protectants (APs) to every extract or (iii) injection prior to GC–MS/MS analysis. Finally, minimization of the matrix effect to the acceptable levels of −20 to 20% for over 80% of investigated pesticides was found when APs mixture was injected at the beginning of the sequence. In this approach, the matrix effects were significantly weaker for some pesticides than when matrix-matched calibration was used.

Compensation for matrix effects in GC analysis of pesticides by using cucumber extract.

Matrix effects (MEs) can adversely affect quantification in pesticide residue analysis using GC. Analyte protectants (APs) can effectively interact with and mask active sites in the GC system, and are added individually or in combination to sample extracts and calibration solutions to minimize errors related to MEs. Unfortunately, APs cannot sufficiently compensate for MEs in all cases. Plant extracts, containing a broad range of natural compounds with AP properties, can also be used for this purpose. In this study, the applicability of cucumber extract as a natural AP mixture was investigated both alone and in combination with traditional APs. Extracts of two selected difficult matrices (onion and garlic) were prepared according to the citrate-buffered QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure. ME values of 40 representative GC-amenable pesticides were compared when calibrating against standards in pure solvent and in cucumber extract, with and without the addition of APs. Using a GC system with a contaminated inlet liner, the use of a cucumber-based calibration solution decreased MEs remarkably. The combination of APs with cucumber raw extract further decreased MEs, resulting in more than 85% of the tested pesticides showing ≤ 10% ME in onion and ≤ 20% ME in garlic. These results demonstrate that the preparation of calibration standards based on cucumber extracts (with or without the addition of APs) is a very useful and practical approach to compensate for MEs in pesticide residue analysis using QuEChERS and GC-MS/MS. The use of various internal standards is furthermore critically discussed.

Quantification of pesticides in food crops using QuEChERS approaches and GC-MS/MS

ABSTRACTThe analysis of pesticides in food products requires accurate measurements for which standardised protocols have been developed. This paper reports the validation of QuEChERS (quick, easy, cheap, effective, rugged, and safe)-based methods applied to three different food samples (brown rice, red pepper and mandarin orange) analysed using gas chromatography-tandem mass spectrometry (GC-MS/MS). The validation of the analytical methods used to measure 113 pesticides, including various insecticides, fungicides and herbicides, was conducted in three food matrices and at three spiking levels by three different laboratories. The set of validated results was interpreted, statistically analysed and visualised using principal component analysis (PCA). Notably, the matrix effects in GC-MS analysis for most of the tested pesticides from different matrices were lowered to practical values (less than 30%) appropriate for routine analysis. This allowed for the quantification of pesticides in the samples using standard and analyte protectants (APs) in the reagent method blank as a screening measure, providing an alternative to matrix-matched calibration. Alternatively, the use of only one standardised calibration (grape matrix) in combination with APs also allowed for the qualitative and semi-quantitative analysis of pesticides in different matrices. The inclusion of APs reduced the differences in the matrix-induced enhancement effects of various samples. The use of an internal standard (IS) to correct the matrix effects was also noted. The normalisation of the analyte peak with triphenyl phosphate (TPP) as an IS resulted in a slight improvement of validation values such as precision and linearity in only one laboratory, but it lowered the value of matrix effects in all three laboratories.

New practical approach for using an analyte protectant for priming in routine gas chromatographic analysis

A new practical approach of using an analyte protectant (AP) for priming a gas chromatography (GC) system has been investigated for routine pesticide residue analysis. Matrix co-extractives are known to cause signal improvement, called the “matrix-induced chromatographic response enhancement effect”. APs are known to provide advantages of signal enhancement independent of the matrix, but it takes time in routine applications to follow the common approach of adding APs to every extract. It is faster and easier to compensate for matrix effects by injecting an AP solution to coat active sites in the GC system at the beginning of the analytical sequence. In this study, 100 pesticides in highly pigmented QuEChERS extracts of chili were evaluated in the AP priming approach using GC–tandem mass spectrometry (MS/MS) over the course of many injections. The AP priming approach presented significantly more consistent results (87% of pesticides met RSD criteria) compared with injection sequences without the use of APs (42% of pesticides met RSD criteria). Also, AP priming with 300 ng d-sorbitol was found to display a similar trend to mixed AP in priming effect. We demonstrated that this approach is fit-for-purpose in routine analysis.

Evaluation of calibration approaches for quantification of pesticide residues in surface water by SPE with small-size cartridges followed by fast GC-MS

A fast GC-MS method for analysis of twenty four GC amenable pesticides in surface water was developed and validated. Extraction of pesticides using solid phase extraction (SPE) with small-size cartridges was performed. Four calibration approaches for quantification purposes, (1) acetonitrile (MeCN) standards, (2) MeCN standards with analyte protectants (APs), (3) matrix-matched standards, and (4) matrix-matched standards with APs, were investigated and statistically compared in terms of validation parameters. Linearity was studied in the concentration range of 0.01–2.5 μg L−1 with coefficients of determination (R2) higher than 0.9466. Satisfactory recoveries at three studied concentration levels (0.05, 0.1 and 1 μg L−1) in the range of 65.6–100.3% with relative standard deviation <19% were obtained. The limits of detection (LODs) and limits of quantification (LOQs) were evaluated for all approaches. LODs ranged from 0.06 to 20 ng L−1 and LOQs from 0.2 to 65 ng L−1. The developed and validated method was applied for the analysis of Danube River water samples.

Evaluation of different calibration approaches in pesticide residues analysis in non-fatty food using fast GC-MS

In the analysis of pesticide residues by GC including GC-MS, adverse effects caused by matrix co-extractives occurs which results in worse precision and trueness of analytical results. Sample extracts of various commodities (fruit and vegetables), prepared by the QuEChERS method were evaluated by gravimetric analysis to compare co-extracted compounds amount. For comparison of matrix background measurement of acetonitrile (MeCN) extracts in full scan mode and SIM monitoring were performed utilising fast GC-MS with quadrupole analyser and narrow-bore columns. In order to evaluate the trueness of quantitative analysis results utilising different calibration standards experiments with synthetic samples (it is a blank sample extract from selected fruit and vegetables–apples, pear, cucumbers and cauliflowers, with the addition of pesticides at the concentration level of 0.05 ng µL−1 (50 µg kg−1)) were performed in this work. This sample represents a simulated real sample, but with the known concentration to check the various calibration approaches utilising calibration standards that would be used in practice: (1) in organic solvent (MeCN) with addition of analyte protectants (APs), (2) matrix-matched standard, (3) matrix-matched standard with addition of APs. For most of studied pesticides very good match of concentration was obtained utilising matrix matched standards (with/without APs). For the majority of the pesticides under study significant overestimation of concentration was observed in acetonitrile standards with APs in all matrices. Repeatability of measurements expressed as relative standard deviations (RSDs) of determined pesticides concentration in all tested matrices was ≤14%.

Fast GC-MS of endocrine disrupting chemicals

An analytical method has been developed and evaluated for the determination of endocrine disrupting chemicals (EDCs)–pesticides in non-fatty food. Twenty-nine analytes of different chemical classes according to EDCs draft list of European Commision were selected. The method is based on fast GC-MS with bench top quadrupole MS detector and electron impact (EI) ionisation (70 eV). For the effective separation programme temperature vaporiser (PTV) injector and narrow-bore column were used. Slightly modified QuEChERS sample preparation technique was applied for the preparation of apple extracts for matrix-matched standards, synthetic sample and real sample extracts. Two internal standards (I.S.)–heptachlor (HPT) and triphenylphosphate (TPP)–were used. The results needed for the method validation were calculated from absolute peak areas and normalised areas to HPT and TPP. The important part of the study is the utilisation of analyte protectants (APs). Three calibrations approaches: matrix-matched standards without/with APs and acetonitrile (MeCN) standards with APs were used. The best results were obtained with matrix-matched standards without APs. The developed and validated analytical method was used for the identification and quantification of EDCs pesticides in real samples (orange, lettuce, strawberry and plum).

Fast gas chromatography for pesticide residues analysis using analyte protectants

Fast GC–MS with narrow-bore columns combined with effective sample preparation technique (QuEChERS method) was used for evaluation of various calibration approaches in pesticide residues analysis. In order to compare the performance of analyte protectants (APs) with matrix-matched standards calibration curves of selected pesticides were searched in terms of linearity of responses, repeatability of measurements and reached limit of quantifications utilizing the following calibration standards in the concentration range 1–500 ng mL −1(the equivalent sample concentration 1–500 μg kg −1): in neat solvent (acetonitrile) with/without addition of APs, matrix-matched standards with/without addition of APs. For APs results are in a good agreement with matrix-matched standards. To evaluate errors of determination of concentration synthetic samples at concentration level of pesticides 50 ng mL −1 (50 μg kg −1) were analyzed and quantified using the above given standards. For less troublesome pesticides very good estimation of concentration was obtained utilizing APs, while for more troublesome pesticides such as methidathion, malathion, phosalone and deltamethrin significant overestimation reaching up to 80% occurred. According to presented results APs can be advantegously used for “easy” pesticides determination. For “difficult” pesticides an alternative calibration approach is required for samples potentially violating MRLs. An example of real sample measurement is shown. In this paper also the use of internal standards (triphenylphosphate (TPP) and heptachlor (HEPT)) for peak areas normalization is discussed in terms of repeatability of measurements and quantitative data obtained. TPP normalization provided slightly better results than the use of absolute peak areas measurements on the contrary to HEPT.