A method for the determination of acrylamide in a broad variety of processed foods by GC–MS using xanthydrol derivatization

A novel GC–MS method was developed for the determination of acrylamide, which is applicable to a variety of processed foods, including potato snacks, corn snacks, biscuits, instant noodles, coffee, soy sauces and miso (fermented soy bean paste). The method involves the derivatization of acrylamide with xanthydrol instead of a bromine compound. Isotopically labelled acrylamide (d ₃-acrylamide) was used as the internal standard. The aqueous extract from samples was purified using Sep-Pak? C₁₈ and Sep-Pak? AC-2 columns. For amino acid-rich samples, such as miso or soy sauce, an Extrelut? column was used for purification or extraction. After reaction with xanthydrol, the resultant N-xanthyl acrylamide was determined by GC–MS. The method was validated for various food matrices and showed good linearity, precision and trueness. The limit of detection and limit of quantification ranged 0.5–5 and 5–20?µg?kg^?1, respectively. The developed method was applied as an exploratory survey of acrylamide in Japanese foods and the method was shown to be applicable for all samples tested.     

Quantitative analysis of acrylamide in tea by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

An effective sample preparation procedure was optimized and a liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the quantitative analysis of acrylamide in tea. [¹³C₃]-acrylamide was used as internal standard. Acrylamide was extracted at 25 °C for 20 min by 10 ml water followed by 10 ml acetonitrile, and then 4 g of magnesium sulfate and 0.5 g of sodium chloride were added to the above mixture under stirring thoroughly. In order to increase the response of acrylamide, 9 ml acetonitrile layer was taken and concentrated to 0.5 ml. Solid-phase extraction with an Oasis MCX cartridge was carried out for clean-up. The limit of detection (LOD) and limit of quantification (LOQ) were 1 and 5 ng/ml, respectively. The recovery efficiency of the extraction procedure ranged between 74% and 79%. The levels of acrylamide in 30 tea samples were less than 100 ng/g. Black, oolong, white and yellow tea samples had quite low acrylamide contents (<20 ng/g). Higher acrylamide levels occurred in baked, roasted, and one sun-dried green tea samples (46–94 ng/g).     

Antioxidant capacity of potato chips and snapshot trends in acrylamide content in potato chips and cereals on the Canadian market

The concentration of acrylamide was measured in selected varieties of five brands of potato chips and breakfast cereals over a 5-year period. Most of the products were purchased in one locality in Canada. Samples were analysed by an isotope dilution (¹³C₃) acrylamide method. They were extracted with water, partitioned with dichloromethane, filtered through a 5 kDa centrifuge filter, cleaned-up on HLB Oasis polymeric and Accucat mixed mode anion and cation exchange SPE columns, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The acrylamide concentration in potato chips varied from 106 to 4630 ng g^?1, while values in cereals varied from 50 to 347 ng g^?1. Wide variations were observed between brands, within brands over time, and between lots of the same brand. A subset of potato chip samples was analysed for in vitro antioxidant activity. No relationship was found between antioxidative capacity of potato chips and their acrylamide content.     

Oleuropein/ligstroside isomers and their derivatives in Portuguese olive mill wastewaters

Olive mill wastewaters (OMW) are a potential source of biophenols, but they have a complex composition with many unknown phenolics. The analysis of purified methanol extracts from two Portuguese OMW by electrospray mass spectrometry in the negative mode showed [M−H]⁻ ions at m/z 539 and m/z 523, corresponding respectively to oleuropein and ligstroside isomers which contain the glucose unit linked to its aromatic moiety. Also, the fragmentation pathway of the [M−H]⁻ ions at m/z 863, 685 and 847 indicated the presence of a diglucoside derivative of the oleuropein isomer and of mono- and diglucosides of the ligstroside isomer, respectively. Moreover, the two OMW samples contained an elenoic derivative of the ion at m/z 685 and a degradation product (m/z 453) of the [M−H]⁻ ion at m/z 523. Future studies focusing on the abundance of these compounds on OMW, as well as their bioactivities, will determine their possible industrial exploitation.     

Validation by collaborative trial of an isotope dilution liquid chromatographic tandem mass spectrometric method to determine the content of acrylamide in roasted coffee

A method for the determination of acrylamide in roasted coffee was subjected to method validation by collaborative trial. The aim was to extend the scope of a method already standardized for the determination of acrylamide in bakery and potato products to include roasted coffee. Modifications of the already standardized method were therefore kept to a minimum. The method was based on aqueous extraction of the roasted coffee matrix and solid-phase extraction (SPE) clean-up followed by isotope dilution high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). The test portion of the sample was spiked with stable isotope-labelled acrylamide and extracted on a mechanical shaker with n-hexane and water for 1 h. The sample extract was centrifuged, the organic phase discarded, and a portion of the aqueous extract further cleaned-up by SPE on an Isolute Multimode cartridge followed by a second clean-up step on an Isolute ENV + cartridge. The volume of the acrylamide-containing fraction eluted from the second SPE column was reduced by evaporation and analysed by LC-MS/MS. Three coffee samples and one aqueous acrylamide standard solution were sent to eleven laboratories from eight European Union Member States. All samples were sent as blind duplicates. Based on the reported results, the relative standard deviations for reproducibility (RSD_R) were 11.5% at a level of 160 µg kg^?1, 10.1% at a level of 263 µg kg^?1, and 9.6% at a level of 585 µg kg^?1. The values for repeatability (RSD_r) in those materials ranged from 1.0% to 3.5%. The method performance parameters satisfied internationally accepted criteria. Hence, the method would be suitable for the enforcement of regulatory limits.     

Screening assay of angiotensin-converting enzyme inhibitory activity from complex natural colourants and foods using high-throughput LC-MS/MS

Inhibition of angiotensin-converting enzyme (ACE) by various foods decreases the blood pressure. ACE inhibitors derived from natural components may be of therapeutic value in preventive medicine. In this study, we report a novel screening assay of ACE inhibitors from complex natural colourants and foods that employ solid phase extraction (SPE), high-throughput liquid chromatography (LC) separation, and stable isotope dilution electrospray tandem mass spectrometry (SID-ESI-MS/MS). When a target sample was subjected to N-Hippuryl-His-Leu (HHL) and ACE in phosphate buffer (pH 7.4), generated hippuric acid (HA) was extracted by SPE. LC/SID-ESI-MS/MS detection of HA allowed us to accurately identify the effects of complex substances such natural colourants and foods that inhibit the ACE of HHL. The major HA and HA-d₅ fragment ions at m/z 180 → 105 and 185 → 110 in the multiple reaction monitoring (MRM) mode can quantify levels that are lower than other methods. The LC/SID-ESI-MS/MS method described here is a rapid, selective, sensitive, and highly reproducible method for the determination of HA in various samples. Based on the assay developed, all samples such as natural colourants, infant formula, soy paste, ketchup, mayonnaise, wheat flour, orange juice, supplement drink, tea, and coffee could be accurately measured for ACE inhibition in various matrices. High-throughput LC/SID-ESI-MS/MS assay has no limitations in the evaluation of inhibition activity in various natural samples such as colour, high-matrix, and processed foods.     

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of glucocorticoid residues in edible tissues of swine,cattle, sheep,and chicken

A confirmatory and quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the presence of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, and fludrocortisone) in the muscles and livers of swine, cattle, and sheep and the muscle of chicken is described. After deconjugation in alkali media, samples were extracted with ethyl acetate for glucocorticoids followed by solid-phase extraction clean-up and reconstitution in the LC mobile phase. The hydrolysis procedure with sodium hydroxide was used to reduce handling time. A single-step solid-phase extraction method was optimized which is suitable for the clean-up of the compounds of interest in many diverse tissue matrices. LC separations were performed on a C₁₈ column with gradient elution using acetonitrile and water (containing 0.2% formic acid) and the two epimers betamethasone and dexamethasone were successfully separated. LC-electrospray ionization (ESI)-MS/MS in negative mode with selected reaction monitoring (SRM) mode was performed to improve method sensitivity and reduce matrix interference. Two SRM transitions were used for each compound. The recovery of glucocorticoids spiked at levels of 0.5–16 µg kg^?1 ranged from 55% to 107%; the between-day relative standard deviations were no more than 15%. The limits of quantification were 0.5–2.0 µg kg^?1 in muscle and 1–4 µg kg^?1 in liver. The optimized procedure was successfully applied to monitor the food at the 2008 Summer Olympics Games in Beijing, China, demonstrating the method to be simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.     

Quantification of main phenolic compounds in sweet and bitter orange peel using CE–MS/MS

The food and agricultural products processing industries generate substantial quantities of phenolics-rich subproducts, which could be valuable natural sources of polyphenols. In oranges, the peel represents roughly 30% of the fruit mass and the highest concentrations of flavonoids in citrus fruit occur in peel. In this work we have carried out the characterisation and quantification of citrus flavonoids in methanolic extracts of bitter and sweet orange peels using CE–ESI–IT–MS. Naringin (m/z 579.2) and neohesperidin (m/z 609.2) are the major polyphenols in bitter orange peels and narirutin (m/z 579.2) and hesperidin (m/z 609.2) in sweet orange peels. The proposed method allowed the unmistakable identification, using MS/MS experiments, and also the quantification of naringin (5.1 ± 0.4 mg/g), neohesperidin (7.9 ± 0.8 mg/g), narirutin (26.9 ± 2.1 mg/g) and hesperidin (35.2 ± 3.6 mg/g) in bitter and sweet orange peels. CE coupled to MS detection can provides structure-selective information about the analytes. In this work we have developed a CE–ESI–IT–MS method for the analysis and quantification of main phenolic compounds in orange peels.     

Liquid chromatography-tandem mass spectrometry determination of N-nitrosamines released from rubber or elastomer teats and soothers

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method as an alternative to a gas chromatography-thermal energy analyser (GC-TEA) method recommended by the European Committee on Standardization (CEN) was validated for the simultaneous determination of eight N-nitrosamines released into artificial saliva from rubber or elastomer teats and soothers. N-nitroso-dipropylamine-d₁₄ (NDPA-d₁₄) was used as internal standard for accurate quantification. The method was validated with relatively good analytical results, including sufficiently low limits of detection (0.1–2?µg?kg^?1 of sample) and good linearity (r ^2?>?0.99) throughout the studied concentration ranges. Intra- and inter-day precisions expressed with the relative standard deviation (RSD, %) were 3.4–8.0% and 4.4–11.3%, which were below the performance criteria based on one-half of the value derived from the Horwitz value. It was also found that the LC-MS/MS method is sufficiently rugged and successfully applicable to its routine analysis for the compliance test of Commission Directive 93/11/EEC.     

Analysis of nisin A,nisin Z and their degradation products by LCMS/MS

The peptides nisin A and nisin Z belong to type-A lantibiotics applied as preservatives in cheese production. The present study optimised and validated a liquid chromatography–tandem mass spectrometry (LCMS/MS) method for the analysis of nisin A in cheese. Since nisin A was not detectable in nisin-containing commercial cheese samples, an additional LCMS/MS method for the quantification of nisin Z was developed and validated. Quantification was performed by external calibration and standard addition. The latter method provided a non-significantly higher recovery rate for the tested cheese matrix. During the production of processed cheese, nisin A and nisin Z undergo significant degradation. Six degradation products of nisin A or nisin Z, respectively, were detected and assigned to nisin A/Z + H₂O, nisin A/Z^1–32, and nisin A/Z^1–32 + H₂O. In two out of eight commercial processed cheese samples, 1.6, resp. 1.7 mg nisin Z/kg cheese was measured, whereas nisin A was not detectable in any of the samples.     

Survey of bottled waters for perchlorate by electrospray ionization mass spectrometry (ESI‐MS) and ion chromatography (IC)

Perchlorate has been identified in ground and surface waters around the USA including some that serve as supplies for drinking water. Because perchlorate salts are used as solid oxidants in rockets and ordnance, water contamination may occur near military or aerospace installations or defense industry manufacturing facilities. This ion has been added to the Environmental Protection Agency's Contaminant Candidate List and the Unregulated Contaminant Monitoring Rule. Concern over perchlorate has prompted many residents in affected areas to switch to bottled water; however, bottled waters have not previously been examined for perchlorate contamination. Should the EPA promulgate a regulation for municipal water systems, US law requires the Food and Drug Administration to take action on bottled water. Methods will therefore be required to determine perchlorate concentrations not only in tap water, but also in bottled waters. Ion chromatography (IC) is the primary technique used for its analysis in drinking water, but it does not provide a unique identification. Confirmation by electrospray ionization mass spectrometry (ESI‐MS) can serve in this capacity. The ESI‐MS method can be applied to these products, but it requires an understanding of matrix effects, especially of high ionic strength that can suppress electrospray. When using methyl isobutyl ketone (MIBK) as the extraction solvent, the ESI‐MS method can reach lower limits of detection of 6 ng ml ⁻¹ for some bottled waters. However, dilution required to negate ionic strength effects in mineral waters can raise this by a factor of 10 or more, depending on the sample. Decyltrimethylammonium cation (added as the bromide salt) is used to produce an ion pair that is extracted into MIBK. After extraction, the sum of the peak areas of the ions C₁₀H₂₁NMe₃(Br)(ClO₄)⁻ (m/z = 380) and C₁₀H₂₁NMe₃(ClO₄)₂⁻ (m/z = 400) is used to quantitate perchlorate. Standard additions are used to account for most of the matrix effects. In this work, eight domestic brands and eight imported brands of bottled water were comparatively analyzed by the two techniques. For comparison, a finished potable water known to contain perchlorate was also tested. None of the bottled waters were found to contain any perchlorate within the lower limit of detection for the IC method. Recoveries on spiked samples subjected to the IC method were ≥98%. Published in 2000 for SCI by John Wiley & Sons, Ltd     

Quantitative determination of mycotoxins in urine by LC-MS/MS

Naturally occurring mycotoxins are responsible for a wide array of adverse health effects. The measurement of urinary mycotoxin levels is a useful means of assessing an individual's exposure, but the development of sensitive and accurate analytical methods for detecting mycotoxins and their metabolites in urine samples is challenging. Urinary mycotoxins are present in low pg ml^–1 concentrations, and the chromatographic identification of their metabolites can be obscured by other endogenous metabolites. We developed an analytical method focused on the selection of two appropriate multiple-reaction monitoring transition for unambiguous identification and quantification of carcinogenic aflatoxin M₁ (AFM₁), ochratoxin A (OTA) and fumonisin B₁, B₂ (FB₁, FB₂) in urine samples from a small volunteer group in a pilot study. AFM₁, OTA, FB₁ and FB₂ were concentrated selectively, interfering substances were removed using an immunoaffinity column (IAC), and mycotoxins were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in combination with a stable-isotope standard-dilution assay (SIDA). The method was sensitive enough to measure mycotoxins and their metabolites at pg ml^–1 levels in urine. The combination of LC-MS/MS and SIDA was critical to distinguishing pseudo-OTα interference from genuine OTα. Twelve urine samples contained OTA ranging from 0.013 to 0.093 ng ml^?1 (mean = 0.031 ng ml^?1). AFM₁ were detected in one sample at a 0.002 ng ml^?1 level, while FB₁ and FB₂ were undetectable in all 12 samples. None of the samples in this pilot study contained a detectable level of OTα, despite the presence of OTA, and this may suggest the need for further epidemiological investigation of OTA exposure in the Korean population.     

Interference-free determination of acrylamide in potato and cereal-based foods by a laboratory validated liquid chromatography–mass spectrometry method

A simple and rapid method was developed and validated for the determination of acrylamide in potato and cereal-based foods by using a single quadrupole liquid chromatography–mass spectrometry (LC–MS) interfaced with positive atmospheric pressure chemical ionization (APCI+). Acrylamide was simply extracted with 0.01 mM acetic acid in a vortex mixer prior to LC–MS analysis. The applicability of validated method was shown for a wide range of processed foods including chips, fries, crisps, breads, biscuits and cookies. The mean recovery was found to be 99.7 with a repeatability of 1.8% in the range 100–1000 ng/g. During LC–MS analyses, the major interfering co-extractive was identified as valine which yields characteristic [M + H]⁺ and compound specific product ions having m/z of 118 and 72, respectively. Valine increased the baseline signal preventing accurate and precise quantitation, and resulted in poorer sensitivity in selected ion monitoring mode. The adverse effect of valine could be limited by instrumentally adjusted delay time or by solid-phase extraction with strong cation-exchanger sorbent.     

Naturally-occurring folates in foods: Method development and analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS)

An accurate method for detecting and quantifying both synthetic (folic acid) and naturally-occurring folates in foods is described. A system capable of analysing the five most commonly occurring folates (pteroylglutamic acid, 5-methyltetrahydrofolate, tetrahydrofolate, 10-formylfolate and 5-formyltetrahydrofolate) in 20 min using liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Quantification of folates was performed using ¹³C labelled internal standards. This paper outlines the development of a comparatively fast LC–MS/MS method, method validation using commercially available folate standards and establishment of the method’s suitability for quantification using selected reaction monitoring (SRM) mass spectrometry. The application of the system was verified by analysing several certified reference materials and comparing results with certified values as determined by microbiological assay. LC–MS/MS promises to be an ideal tool for the quantitative analysis of folates in food.     

Determination of aflatoxin M1 in milk by triple quadrupole liquid chromatography-tandem mass spectrometry

A sensitive and reliable method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an electrospray-positive ionization method was developed for the determination of aflatoxin M₁ in milk. This method includes simple extraction of the sample with acetonitrile by ultrasonic, separation on an MGIII-C₁₈ column using 0.01% formic acid buffer/acetonitrile (60 : 40, v/v) as mobile phase, and MS/MS detection using multiple reaction-monitoring mode. Average recoveries of aflatoxin M₁ from spiked samples at concentrations of 0.02 and 1 ng ml^?1 ranged from 77% to 94%, with a 6% relative standard deviation. The limit of detection and limit of quantification were 0.006 and 0.02 ng ml^?1, respectively. The standard curve was linear between 0.02 and 20.0 ng ml^?1. The recommended method is simple, rapid, specific and reliable for the routine monitoring of aflatoxin M₁ in milk.     

Determination of chloramphenicol in shrimp by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS)

A liquid chromatographic method with electrospray ionization tandem mass spectrometric (LC-ESI-MS-MS) detection and identification is described for the determination of chloramphenicol (CAP) in shrimp tissue. Homogenized shrimp samples were extracted by liquid–liquid extraction using ethyl acetate. d₅-Chloramphenicol (5D-CAP) was used as the internal standard. Data acquisition was in negative-ion multiple reaction monitoring (NMRM) mode using three transition reactions for CAP (m/z 321?→?152, m/z 321?→?257 and m/z 321?→?194) and two for d₅-chloramphenicol (m/z 326?→?262 and m/z 326?→?157). Method validation was carried out according to European Commission decision 2002/657/EC. The calibration curve was linear in the range 0.10–2.00 µg l^?1, with typical r ²?>?0.99. The decision limit (CCα) and detection capability (CCβ) were 0.06 and 0.10 µg kg^?1, respectively. There was no influence of the matrix on the determination of chloramphenicol.     

Acrylamide in Caribbean foods – Residual levels and their relation to reducing sugar and asparagine content

The acrylamide levels in commercial and homemade Caribbean foods were determined by pre-derivatisation of acrylamide to 2-bromopropenamide and analysed by gas chromatography with mass spectrometric (GC/MS) detection. Over 100 Caribbean food samples were analysed for the presence of acrylamide. These samples include: biscuits, breakfast cereals, banana chips and home-prepared foods: breadfruit; Artocarpus altilis, banana fritters, and dumplings. The limit of detection (LOD) for the GC/MS method was found to be dependent on the type of column used for the GC/MS analysis. The DB-1701 and the DB-VRX columns gave LODs of 20 and 4 μg/kg, respectively. Acrylamide has not been found in raw foods or foods which have been cooked by boiling. Its content in all other foods had concentrations in the range, 65–3640 μg/kg. The relationship between acrylamide levels and precursor concentration as well as the health implications of our findings are discussed.     

固相萃取-高效液相色谱-串联质谱法测定食品中的丙烯酰胺

建立高效液相色谱-串联质谱仪测定食品中丙烯酰胺含量的方法。以[D3]-丙烯酰胺为同位素内标,使用20mg/mL淀粉酶溶液对样品进行酶解,经水浴、除脂、离心,利用固相萃取柱进行净化并浓缩后,通过高效液相色谱-串联质谱仪以多选择反应监测模式进行分析测定。该方法的检出限和定量限分别为4.8μg/kg和16.5μg/kg。本方法具有操作方便、准确率高、重复性好等优点,可广泛应用于各类食品中丙烯酰胺含量的检测。     

Pyrrolizidine alkaloids in honey: comparison of analytical methods

Pyrrolizidine alkaloids (PAs) are a structurally diverse group of toxicologically relevant secondary plant metabolites. Currently, two analytical methods are used to determine PA content in honey. To achieve reasonably high sensitivity and selectivity, mass spectrometry detection is demanded. One method is an HPLC-ESI-MS-MS approach, the other a sum parameter method utilising HRGC-EI-MS operated in the selected ion monitoring mode (SIM). To date, no fully validated or standardised method exists to measure the PA content in honey. To establish an LC-MS method, several hundred standard pollen analysis results of raw honey were analysed. Possible PA plants were identified and typical commercially available marker PA-N-oxides (PANOs). Three distinct honey sets were analysed with both methods. Set A consisted of pure Echium honey (61–80% Echium pollen). Echium is an attractive bee plant. It is quite common in all temperate zones worldwide and is one of the major reasons for PA contamination in honey. Although only echimidine/echimidine-N-oxide were available as reference for the LC-MS target approach, the results for both analytical techniques matched very well (n?=?8; PA content ranging from 311 to 520?µg?kg^?1). The second batch (B) consisted of a set of randomly picked raw honeys, mostly originating from Eupatorium spp. (0–15%), another common PA plant, usually characterised by the occurrence of lycopsamine-type PA. Again, the results showed good consistency in terms of PA-positive samples and quantification results (n?=?8; ranging from 0 to 625?µg?kg^?1 retronecine equivalents). The last set (C) was obtained by consciously placing beehives in areas with a high abundance of Jacobaea vulgaris (ragwort) from the Veluwe region (the Netherlands). J. vulgaris increasingly invades countrysides in Central Europe, especially areas with reduced farming or sites with natural restorations. Honey from two seasons (2007 and 2008) was sampled. While only trace amounts of ragwort pollen were detected (0–6.3%), in some cases extremely high PA values were detected (n?=?31; ranging from 0 to 13019?µg?kg^?1, average?=?1261 or 76?µg?kg^?1 for GC-MS and LC-MS, respectively). Here the results showed significantly different quantification results. The GC-MS sum parameter showed in average higher values (on average differing by a factor 17). The main reason for the discrepancy is most likely the incomplete coverage of the J. vulgaris PA pattern. Major J. vulgaris PAs like jacobine-type PAs or erucifoline/acetylerucifoline were not available as reference compounds for the LC-MS target approach. Based on the direct comparison, both methods are considered from various perspectives and the respective individual strengths and weaknesses for each method are presented in detail.     

Pressurised liquid extraction combining LC–DAD–ESI/MS analysis as an alternative method to extract three major flavones in Citrus reticulata ‘Chachi’ (Guangchenpi)

Pressurised liquid extraction (PLE) of three major flavones (hesperidin, nobiletin, and tangeretin) from the peels of Citrus reticulata ‘Chachi’ (Guangchenpi) was investigated. These flavones were quantified and analysed by liquid chromatography–diode array detector–electrospray ionisation mass spectrometry (LC–DAD–ESI/MS). The PLE procedure was optimised, validated and compared with other conventional extraction techniques. PLE gained the best result due to the highest extraction efficiency within the shortest extraction time. The optimal conditions of PLE were employing 70% methanol as extraction solvent at a temperature of 160 °C and extraction pressure of 1500 psi, using one extraction cycle with a static extraction time of 20 min. MS coupling with an ESI interface in the positive ion mode was used as the detection technique. This is the first report on combining PLE with LC–DAD–ESI/MS for the extraction and quantification of flavones in Citrus reticulata ‘Chachi’.