Optimization of SPME-Arrow-GC/MS Method for Determination of Free and Bound Volatile Organic Compounds from Grape Skins.

(1) Background: Solid phase microextraction (SPME)-Arrow is a new extraction technology recently employed in the analysis of volatiles in food materials. Grape volatile organic compounds (VOC) have a crucial role in the winemaking industry due to their sensory characteristics of wine.; (2) Methods: Box-Behnken experimental design and response surface methodology were used to optimise SPME-Arrow conditions (extraction temperature, incubation time, exposure time, desorption time). Analyzed VOCs were free VOCs directly from grape skins and bound VOCs released from grape skins by acid hydrolysis.; (3) Results: The most significant factors were extraction temperature and exposure time for both free and bound VOCs. For both factors, an increase in their values positively affected the extraction efficiency for almost all classes of VOCs. For free VOCs, the optimum extraction conditions are: extraction temperature 60 °C, incubation time 20 min, exposure time 49 min, and desorption time 7 min, while for the bound VOCs are: extraction temperature 60 °C, incubation time 20 min, exposure time 60 min, desorption time 7 min.; (4) Conclusions: Application of the optimized method provides a powerful tool in the analysis of major classes of volatile organic compounds from grape skins, which can be applied to a large number of samples.

[Detection of Residual Solvents in Commercial Supplements Using Headspace (HS)-GC-MS].

In order to understand the actual state of residual solvents contained in commercial supplements, we performed a simultaneous analysis of residual solvents by headspace (HS)-GC-MS with reference to the Japanese Pharmacopoeia's "Residual Solvents", for 29 products selected from among commercial supplements (e.g., revitalizers, weight loss pills) that are deeply colored or contain coating agents and extract powder. As a result, benzene (class 1) was detected in eight black-colored supplements, and hexane (class 2B) was also detected in one of those products. On the other hand, methanol (class 2A) was detected in four products containing coating agents and extract powders, such as citrus peel extract. None of these residual solvents exceeded the concentration limits set by the Japanese Pharmacopoeia. Benzene was detected at 1.7 µg/g, which was near the concentration limit, in some products. As raw materials used for the manufacture of the black-colored supplements from which benzene was detected commonly included activated carbon, we analyzed the residual solvents contained in activated carbon commercially available for use as food additive and in food production and medicine. As a result, benzene was detected at high concentrations in activated carbon made from hemp (approximately 29 µg/g) and bamboo (approximately 140 µg/g).

Thermal degradation of metabolites in urine using multiple isotope-labelled internal standards for off-line GC metabolomics - effects of injector and oven temperatures.

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). An optimized derivatization protocol has been successfully applied using multiple isotope labelled analytical internal standards of selected deuterated and 13C selected compounds, covering a range of different groups of metabolites for non-automated GC metabolomics (off-line). Moreover, the study was also realized in a pooled urine sample, following metabolic profiling. A study of thermal degradation of metabolites due to GC inlet and oven programs (fast, slow) was performed, where the results indicated that both GC oven programs (fast and slow) negatively affected the thermal stability of the metabolites, while the fast-ramp GC program also suppressed MS signals. However, the use of multiple internal standards can overcome this drawback. The application of extended temperature ramp GC program presented identical behaviour on metabolite stability and better chromatographic separation combined with much lower signal suppression, compared to a short temperature ramp program. No effects were observed for organic acids, fatty acids, sugars and sugar alcohols, while significant differences were observed for amino acids. GC metabolomics is a strong tool that can facilitate analysis, but special attention is required for sampling handling and heating, before and during the GC analysis. The use and application of multiple multi-group internal standards is highly recommended.

[Examination of Analytical Method for Mothproofing Agents to Revise the Official Methods Based on "Act on the Control of Household Products Containing Harmful Substances"].

In Japan, the use of mothproofing agents [dieldrin and 4,6-dichloro-7-(2,4,5-trichlorophenoxy)-2-trifluoromethylbenzimidazole; DTTB] in textiles is regulated by the Act on the Control of Household Products Containing Harmful Substances. Since official analytical methods for these agents have been in place for approximately 40 years, we developed an improved method in a previous study. In the present study, we validated this method. Accordingly, six laboratories analyzed the sample prepared at 3 µg/g (1/10 of the regulation value) and 30 µg/g (the regulation value). The high accuracy of the results for these samples in almost all the cases (accuracy: 70-120%, repeatability: <10%, reproducibility: <15%), confirmed the validity of the method. In addition, we examined three samples that were distributed before the introduction of the regulation. The analysis results for these samples showed little variation between laboratiories, indicating that our method is also applicable to actual samples. Meanwhile, the quantitative value was clearly lower in one laboratory than in the others. We presumed that the enhanced effect of the sample matrix (matrix effect) on the internal standards in GC-MS analysis was the main cause for this trend. Therefore, we examined the analytical method using polyethylene glycol 300 (PEG) as an analyte protectant. As PEG minimized the GC-MS response difference between the standard solution and the matrix-containing solution, GC-MS analysis with PEG would be useful for matrix effect measurements in this method.

Determination of Saffron Volatiles by HS-SBSE-GC in Flavored Cured Ham.

At present, the development of new agri-food products, including flavored meat products presented in ready-to-eat vacuum packs, is encouraged. The addition of ingredients used as flavoring agents creates the need to be able to determine the volatile compounds responsible for their characteristic aroma. The aim of this study is to propose, develop, and validate a new method that uses headspace-stir bar sorptive extraction-gas chromatography/mass spectrometry (HS-SBSE-GC/MS) to determine the saffron aroma in cured ham flavored with this spice. Results showed that safranal was the main volatile compound that could be identified and quantified in cured ham flavored with saffron. This analytical method was adequate in terms of linearity, selectivity, sensitivity, and accuracy. To our knowledge, this is the first time that an HS-SBSE-GC/MS method for determining the saffron aroma of flavored cured ham has been developed and validated, and it is of interest to agri-food industries.

A Comparison of the Composition of Selected Commercial Sandalwood Oils with the International Standard.

Sandalwood oils are highly desired but expensive, and hence many counterfeit oils are sold in high street shops. The study aimed to determine the content of oils sold under the name sandalwood oil and then compare their chromatographic profile and α- and ß santalol content with the requirements of ISO 3518:2002. Gas chromatography with mass spectrometry analysis found that none of the six tested "sandalwood" oils met the ISO standard, especially in terms of α-santalol content. Only one sample was found to contain both α- and ß-santalol, characteristic of Santalum album. In three samples, valerianol, elemol, eudesmol isomers, and caryophyllene dominated, indicating the presence of Amyris balsamifera oil. Another two oil samples were found to be synthetic mixtures: benzyl benzoate predominating in one, and synthetic alcohols, such as javanol, polysantol and ebanol, in the other. The product label only gave correct information in three cases: one sample containing Santalum album oil and two samples containing Amyris balsamifera oil. The synthetic samples described as 100% natural essential oil from sandalwood are particularly dangerous and misleading to the consumer. Moreover, the toxicological properties of javanol, polysantol and ebanol, for example, are unknown.

A chiral GC-MS method for analysis of secondary amino acids after heptafluorobutyl chloroformate & methylamine derivatization.

L-amino acids (L-AAs) play different important roles in the physiology of all living organisms. Their chiral counterparts, D-amino acids (D-AAs) are increasingly being recognized as essential molecules in many biological systems. Secondary amino acids with cyclic structures, such as prolines, exhibit conformational rigidity and thus unique properties in the structural and protein folding. Despite their widespread occurrence, much less attention was paid to their chiral analysis, particularly when the minor, typically D-enantiomer, is present in low amounts in a complex biological matrix. In this paper, a cost-effective, chiral GC-MS method is described for capillary Chirasil-L-Val separation of nine cyclic secondary amino acid enantiomers with four-, five-, and six-membered rings, involving azetidine-2-carboxylic acid, pipecolic acid, nipecotic acid, proline, isomeric cis/trans 3-hydroxy, 4-hydroxyproline, and cis/trans-5-hydroxy-L-pipecolic acid in the excess of its enantiomeric antipode. The sample preparation involves in-situ derivatization with heptafluorobutyl chloroformate, simultaneous liquid-liquid micro-extraction into isooctane followed by amidation of the arising low-polar derivatives with methylamine, an evaporation step, re-dissolution, and final GC-MS analysis. The developed method was used for analyses of human biofluids, biologically active peptides containing chiral proline constituents, and collagen.

A perspective on the standards describing mass spectrometry-based metabolic phenotyping (metabolomics/metabonomics) studies in publications.

As metabolic phenotyping (metabolomics, metabonomics and also lipidomics) gains in popularity and new investigators enter the field, the need to maintain and improve standards in publication is ever more pressing. In this perspective the requirements for information that should be included in manuscripts published in the Journal of Chromatography B, to ensure that the work is both credible and repeatable, are discussed. These include aspects such as study design, ethics, quality assurance (QA), quality control (QC) and data processing. In addition, aspects such as the level of confidence required for reporting metabolite identification (to a level where they could be subsequently used to develop hypotheses) are discussed.

Improving Quantification of tabun, sarin, soman, cyclosarin, and sulfur mustard by focusing agents: A field portable gas chromatography-mass spectrometry study.

Commercial gas chromatograph-mass spectrometers, one of which being Inficon's HAPSITE® ER, have demonstrated chemical detection and identification of nerve agents (G-series) and blistering agents (mustard gas) in the field; however most analyses relies on self-contained or external calibration that inherently drifts over time. We describe an analytical approach that uses target-based thermal desorption standards, called focusing agents, to accurately calculate concentrations of chemical warfare agents that are analyzed by gas chromatograph-mass spectrometry. Here, we provide relative response factors of focusing agents (2-chloroethyl ethyl sulfide, diisopropyl fluorophosphate, diethyl methylphosphonate, diethyl malonate, methyl salicylate, and dichlorvos) that are used to quantify concentrations of tabun, sarin, soman, cyclosarin and sulfur mustard loaded on thermal desorption tubes (Tenax® TA). Aging effects of focusing agents are evaluated by monitoring deviations in quantification as thermal desorption tubes age in storage at room temperature and relative humidity. The addition of focusing agents improves the quantification of tabun, sarin, soman, cyclosarin and sulfur mustard that is analyzed within the same day as well as a 14-day period. Among the six focusing agents studied here, diisopropyl fluorophosphate has the best performance for nerve agents (G-series) and blistering agents (mustard gas) compared to other focusing agents in this work and is recommended for field use for quantification. The use of focusing agent in the field leads to more accurate and reliable quantification of Tabun (GA), Sarin (GB), Soman (GD), Cyclosarin (GF) and Sulfur Mustard (HD) than the traditional internal standard. Future improvements on the detection of chemical, biological, radiological, nuclear, and explosive materials (CBRNE) can be safely demonstrated with standards calibrated for harmful agents.

Interpretation of omics data analyses.

Omics studies attempt to extract meaningful messages from large-scale and high-dimensional data sets by treating the data sets as a whole. The concept of treating data sets as a whole is important in every step of the data-handling procedures: the pre-processing step of data records, the step of statistical analyses and machine learning, translation of the outputs into human natural perceptions, and acceptance of the messages with uncertainty. In the pre-processing, the method by which to control the data quality and batch effects are discussed. For the main analyses, the approaches are divided into two types and their basic concepts are discussed. The first type is the evaluation of many items individually, followed by interpretation of individual items in the context of multiple testing and combination. The second type is the extraction of fewer important aspects from the whole data records. The outputs of the main analyses are translated into natural languages with techniques, such as annotation and ontology. The other technique for making the outputs perceptible is visualization. At the end of this review, one of the most important issues in the interpretation of omics data analyses is discussed. Omics studies have a large amount of information in their data sets, and every approach reveals only a very restricted aspect of the whole data sets. The understandable messages from these studies have unavoidable uncertainty.

A selective method for quantification of diazinon in human plasma by GC-MS.

Diazinon is a synthetic molecule known as an organophosphorus insecticide. It is used for gardens and for agriculture. This molecule represents a toxicological concern for humans. For this reason, the detection and the quantification of the diazinon in human samples are important in order to monitor an exposure and to diagnose intoxications. The aim of this work is to develop a selective method for the quantification of diazinon in human plasma by means of a gas chromatography (GC) with a mass spectroscopy (MS) detector. The method presented in this article includes a liquid-liquid extraction (LLE) using dichloromethane/propanol-2/heptane. The correlation coefficient for the calibration curve is 0.992. The limit of detection (LOD) and the limit of quantification (LOQ) are respectively 5 µg/L and 2 µg/L. The average recovery of the three different concentrations (15, 250 and 375 µg/L) varies from 22.8% to 31%. A test of stability at ambient temperature for 24 hours and at 4 °C for 48 hours was conducted. The relative error factor was found to be inferior to 15%. The results obtained show that the method developed gives satisfying validation parameters in human plasma. Therefore, the compound could be detected at very low concentration with a good linearity. This method can be used with other matrices, such as blood or urine, for a partial validation.

[Recommendations for urinary organic acids analysis]. / Recommandations concernant l'analyse des acides organiques urinaires.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Organic acid chromatography allows the identification of several hundred compounds and the quantification of the main molecules of interest. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from the French society for inborn errors of metabolism (SFEIM) recommends an approach to accredit organic acid chromatography. Validation parameters and recommendations are discussed in this specific framework.

LC-MS/MS-based in vitro and in vivo investigation of blood-brain barrier integrity by simultaneous quantitation of mannitol and sucrose.

BACKGROUND: Understanding the pathophysiology of the blood brain-barrier (BBB) plays a critical role in diagnosis and treatment of disease conditions. Applying a sensitive and specific LC-MS/MS technique for the measurement of BBB integrity with high precision, we have recently introduced non-radioactive [13C12]sucrose as a superior marker substance. Comparison of permeability markers with different molecular weight, but otherwise similar physicochemical properties, can provide insights into the uptake mechanism at the BBB. Mannitol is a small hydrophilic, uncharged molecule that is half the size of sucrose. Previously only radioactive [3H]mannitol or [14C]mannitol has been used to measure BBB integrity. METHODS: We developed a UPLC-MS/MS method for simultaneous analysis of stable isotope-labeled sucrose and mannitol. The in vivo BBB permeability of [13C6]mannitol and [13C12]sucrose was measured in mice, using [13C6]sucrose as a vascular marker to correct for brain intravascular content. Moreover, a Transwell model with induced pluripotent stem cell-derived brain endothelial cells was used to measure the permeability coefficient of sucrose and mannitol in vitro both under control and compromised (in the presence of IL-1ß) conditions. RESULTS: We found low permeability values for both mannitol and sucrose in vitro (permeability coefficients of 4.99 ± 0.152 × 10-7 and 3.12 ± 0.176 × 10-7 cm/s, respectively) and in vivo (PS products of 0.267 ± 0.021 and 0.126 ± 0.025 µl g-1 min-1, respectively). Further, the in vitro permeability of both markers substantially increased in the presence of IL-1ß. Corrected brain concentrations (Cbr), obtained by washout vs. vascular marker correction, were not significantly different for either mannitol (0.071 ± 0.007 and 0.065 ± 0.009 percent injected dose per g) or sucrose (0.035 ± 0.003 and 0.037 ± 0.005 percent injected dose per g). These data also indicate that Cbr and PS product values of mannitol were about twice the corresponding values of sucrose. CONCLUSIONS: We established a highly sensitive, specific and reproducible approach to simultaneously measure the BBB permeability of two classical low molecular weight, hydrophilic markers in a stable isotope labeled format. This method is now available as a tool to quantify BBB permeability in vitro and in vivo in different disease models, as well as for monitoring treatment outcomes.

Excretion of 19-norandrosterone after consumption of boar meat.

The consumption of the offal of noncastrated pigs can lead to the excretion of 19-norandrosterone (NorA) in urine of humans. In doping control, GC/C/IRMS is the method of choice to differentiate between an endogenous or exogenous origin of urinary NorA. In some cases, after the consumption of wild boar offal, the δ13 C values of urinary NorA fulfill the criteria of an adverse analytical finding due to differing food sources of boar and consumer. However, consumption of wild boar's offal is not very common in Germany, and thus, the occurrence of such an analytical finding is unlikely. In contrast, the commerce with wild boar meat has increased in Germany within the last years. Up to 20,000 tons of wild boar meat are annually consumed. In order to probe for the probability of the occurrence of urinary NorA after consumption of wild boar meat, human urine samples were tested following the ingestion of commercially available game. In approximately half of the urine samples, traces of NorA were detected postadministration of 200 to 400 g boar meat. The highest urinary concentration was 2.9 ng/ml, and significant amounts were detected up to 9 h after the meal. δ13 C values ranged from -18.5‰ to -23.5‰, which would have led to at least two adverse analytical findings if the samples were collected in an antidoping context. IRMS analysis on German boar tissue samples showed that δ13 C values for wild boar's steroids are unpredictable and may vary seasonally.

Alternative markers for Methylnortestosterone misuse in human urine.

Methylnortestosterone is a progestin and synthetic androgenic anabolic steroid, prohibited by WADA. Methylnortestosterone misuse is commonly detected by monitoring the parent compound and its main metabolites, 17α-methyl-5α-estrane-3α, 17ß-diol (M1) and 17α-methyl-5ß-estrane-3α, 17ß-diol (M2), in the glucuronide fraction. In the current study, a direct detection of methylnortestosterone sulfo-conjugated metabolites after ethyl acetate extraction and analysis by LC/Q/TOF-MS in negative ionization mode was performed, detecting two main sulfate metabolites (S1, S2). For the characterization of metabolites, samples from the excretion study, were additionally analyzed by GC-MS, after solvolysis and per TMS derivatization. RT and MS data collected, were compared with RT and MS data from metabolites of 17z-methyl-5α/ß-estrane-3α/ß, 17z-diols structures with prefixed stereochemistry at 3 and 5 positions, synthesized through Grignard reaction from 19-noretiocholanolone, 19-norandrosterone and 19-norepiandrosterone. Confirmed sulfate metabolites were S1, 17α-methyl-5α-estrane-3α, 17ß-diol 3α sulfate (detected up to 72 h) and S2, 17α-methyl-5ß-estrane-3α, 17ß-diol 3α sulfate (detected up to 192 h). Furthermore, applying targeted analysis based on RT and MS data of the synthesized metabolites two additional metabolites M3, 17ß-methyl-5ß-estrane-3α, 17α-diol and M4, 17ß-methyl-5α-estrane-3α, 17α-diol were detected in the glucuronide fraction and one more metabolite (S3) 17ß-methyl-5ß-estrane-3α, 17α-diol was detected in the sulfate fraction in lower abundance until the end of the excretion study (192 h). Interestingly, S2 could also be detected after the direct analysis of non-hydrolyzed steroid by GC-MS/MS as artifact, following normal ProcIV anabolic steroid procedure and using diethylether as extraction solvent.

Evaluation of epiandrosterone as a long-term marker of testosterone use.

Identification and evaluation of long-term markers is crucial in prolonging the detection window for anabolic steroid abuse in sport. Recently, sulfoconjugated epiandrosterone was identified as a potential long-term marker for the abuse of certain endogenous anabolic agents, including testosterone, which continues to be widely used as a performance enhancing agent in sport. To evaluate the applicability of epiandrosterone sulfate as a marker for testosterone use, administration studies were conducted with multiple modes of testosterone administration - transdermal, intramuscular, and subcutaneous. A modified sample preparation method was used to collect both glucuronidated and sulfoconjugated analytes of interest. Carbon isotope ratio measurements from the administration studies are presented here. Epiandrosterone was less effective than the conventionally used target compounds for detection of the low dose application (transdermal gel). With intramuscular administration, epiandrosterone was more diagnostic than with transdermal administration, but it did not prolong the detection window more than the conventional target compounds. With subcutaneous administration, the doses administered to the subjects were varied and the effect on the epiandrosterone values was dependent on the magnitude of the dose administered. Epiandrosterone does not appear to be a useful marker in the detection of low dose testosterone administration. It is responsive to higher dose administration, but it does not provide an extension of the detection window relative to conventional target compounds.

Carbon isotopic characterization of prednisolone and prednisone pharmaceutical formulations: Implications in antidoping analysis.

Twenty-two pharmaceutical formulations containing prednisolone or prednisone commercially available in Italy, Belgium, Spain, Brazil, and India were analyzed through a specific gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) method. All of them showed typical non-endogenous δ13 C values, except for the Belgian nasal spray, Sofrasolone®, with a less depleted 13 C content (-17.84 ± 0.18‰). Observational studies were performed on two volunteers in therapy with Sofrasolone® to confirm the applicability of the method and to suggest adequate interpretation criteria also in the case of drugs with less negative δ13 C values. Urine samples were collected before, during, and within the 36 hours after the administration of the spray. Both δ13 C values and urinary concentrations of prednisolone and prednisone were evaluated. All samples were subjected to an adequate pre-treatment (enzymatic hydrolysis, liquid/liquid extraction, and two sequential HPLC steps) before injection to the GC-C-IRMS instrument, according to the method recently developed and validated in our laboratory. Pregnanediol (PD), tetrahydro-11-deoxycortisol (THS), and pregnanetriol (PT) were selected as endogenous reference compounds (ERC). The excretion profile was estimated through liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method used routinely for the quali-quantitative detection of glucocorticoids. δ13 C values and urinary levels of prednisolone and prednisone were also determined after the intake of one single vial of Sintredius®, a prednisolone oral formulation with a conventional more negative δ13 C value (-29.28 ± 0.25‰). Finally, the potential masking effect that combined therapy with Sofrasolone® and Sintredius® could induce on the IRMS findings was investigated.

The underlying challenges that arise when analysing short-chain chlorinated paraffins in environmental matrices.

As short-chain chlorinated paraffins (SCCPs) are listed on several monitoring programs, validated methods are essential. However, their complexity and the lack of commercially available certified reference materials (RMs) hinder a proper validation of methods. Instead, one method is usually 'validated' by evaluating performances and results of spiked materials with that of one other method, which could easily lead to unreliable results. This study evaluated four analytical methods with different principles (i.e. comprehensive two dimensional GC coupled to a micro electron capture detector, developed for this study, chloride enhanced atmospheric pressure chemical ionization triple quadrupole time of flight MS (APCI-QToF-HRMS), GC coupled to an electron capture negative ion low resolution MS (GC-ECNI-LRMS) and carbon skeleton GC-MS), investigated the comparability in SCCP determination in spiked and naturally contaminated samples and determined SCCP amounts in candidate RMs for possible certification. The results cast doubt on the use of the most commonly applied method (i.e. GC-ECNI-LRMS), as well as using spiked materials for method validation. The APCI-QToF-HRMS method was found most promising as it achieves the required MS resolution (>21,000), is relatively fast and can detect also other CPs. The suitable identified SCCP levels in the candidate RMs and the agreement in results between the methods bring the first certification of a RM for SCCPs within reach.

Development of candidate reference method for accurate determination of four polycyclic aromatic hydrocarbons in olive oil via gas chromatography/high-resolution mass spectrometry using 13C-labeled internal standards.

We developed an isotope-dilution gas chromatography/high-resolution mass spectrometry (ID-GC/HRMS) method for the accurate determination of four polycyclic aromatic hydrocarbons (PAHs) in olive oil. The clean-up steps were optimized to sufficiently remove co-extracted oil matrix using the EZ-POP NP dual-layer and NH2 solid-phase extraction (SPE) cartridges. However, the cleaned sample still contained some matrix residues, which caused a bias. When 13C-labeled PAHs were used as the internal standards instead of deuterated PAHs, the recovery results improved in both low- and high-resolution MS conditions. Furthermore, the HRMS analysis facilitated to obtain more accurate results. The accuracy and precision of the optimized ID-GC/HRMS method were validated using PAH-fortified (0.5, 3, and 6 µg/kg) olive oil. The recoveries and relative standard deviations obtained for all the PAHs/levels were 97.5-102% and ~1%, respectively. Measurement uncertainties were generally within 5% (with a 95% confidence level).

Analysis of organophosphorus and pyrethroid pesticides in organic and conventional vegetables using QuEChERS combined with dispersive liquid-liquid microextraction based on the solidification of floating organic droplet.

A simple, sensitive and environmentally-friendly method for determining organophosphorus and pyrethroid pesticides in vegetables was developed to better evaluate the risk of consuming them. The pesticides in vegetables were extracted, purified and concentrated by using the QuEChERS (quick, easy, cheap, effective, rugged and safe method) combined DLLME-SFO (dispersive liquid-liquid microextraction based on solidification of floating organic droplet) techniques. The key parameters were optimized through orthogonal array experimental design and statistical analysis. The linearity of the calibration curves was satisfied in matrix-matched standard solution with R2 ≥ 0.99. The limits of detection and limits of quantification were 0.3-1.5 and 0.9-4.7 µg/kg, respectively. The average recoveries of pesticides were 61.6-119.4% with relative standard deviations <16.1%. Furthermore, the method was applied successfully to analyse the pesticides in 15 pairs of organic and conventional vegetables. These results reflect the efficiency, reliability and robustness of the developed method.