Single-laboratory validation of a gas chromatography-mass spectrometry method for quantitation of 15 European priority polycyclic aromatic hydrocarbons in spiked smoke flavourings

An existing method was adapted to the purpose and validated in-house according to the IUPAC harmonised guideline for the determination of 15 EU priority polycyclic aromatic hydrocarbons (PAHs) in primary smoke condensates (PSCs) that are used to produce smoke flavourings for human consumption. Limits of detection (LOD) varied between 0.1 and 1.3 microg/kg, limits of quantitation (LOQ) between 0.5 and 4 microg/kg for the various PAHs in PSCs. The coefficient of variance of the repeatability was between 0.7% (benzo[a]pyrene) and 30% (dibenzo[a,h]pyrene) relative standard deviation, depending on the analyte. The recoveries varied between 100 and 102% (dibenzo[a,l]pyrene) and 69-83% (dibenzo[a,h]pyrene) over the analytical range of 5-35 microg/kg.     

Analysis of EU priority polycyclic aromatic hydrocarbons in food supplements using high performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector

High performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector (HPLC/UV-FLD) has been used to set up a method to detect the 15(+1) EU priority polycyclic aromatic hydrocarbons (PAHs) in food supplements covering the categories of dried plants and plant extracts excluding oily products. A mini validation was performed and the following parameters have been determined: limit of detection, limit of quantification, precision, recovery and linearity. They were in close agreement with quality criteria described in the Commission Regulation (EC) No 333/2007 concerning the PAH benzo[a]pyrene in foodstuffs, except the not fluorescent cyclopenta[c,d]pyrene for which the UV detection leads to a higher limit of detection. Analysis of twenty commercial food supplements covering mainly the class of dried plants was performed to evaluate their PAHs contamination levels and to test the applicability of the method to various plant matrices. Fifty percent of analyzed samples showed concentration exceeding 2 μg kg⁻¹ for one or more PAHs.     

Innovative method for determination of 19 polycyclic aromatic hydrocarbons in food and oil samples using gas chromatography coupled to tandem mass spectrometry based on an isotope dilution approach

An efficient and selective analytical method for the determination and the quantification of 19 polycyclic aromatic hydrocarbons (PAHs) in food and oil has been developed. This method includes the monitoring of 15 PAHs stated as a priority by the EU in their 2005/108 recommendation. The samples were extracted according to a selective extraction step using pressurized liquid extraction followed by a purification with polystyrene-divinylbenzene SPE. Identification and quantification were performed using GC-MS/MS, with an isotope dilution approach using (13)C-labelled PAHs. The novel combination of selective extraction followed by purification provides highly purified analytes combined to a fast and automated method. The advantages of GC-MS/MS as compared to other detection methods are tremendous in terms of sensitivity, selectivity and interpretation facilities. Limits of detection varied between 0.008 and 0.15 microg kg(-1), limits of quantification between 0.025 and 0.915 microg kg(-1) for PAHs in food. The calibration curves showed a good linearity for all PAHs (R(2)>0.99) and precision and recovery were fit for purpose. Trueness of the method was carried out using the US National Institute of Standards and Technology SRM 2977 reference material.     

Rapid SPE-HPLC determination of the 16 European priority polycyclic aromatic hydrocarbons in olive oils

Polycyclic aromatic hydrocarbons (PAHs) are a large class of organic compounds. It has been established that the main source of exposure to these compounds for human beings is through food, particularly fats and oils, due to the lipophilic nature of these polycyclic compounds. The aim of this work was to optimise and validate a method involving SPE and HPLC for rapid determination of the 16 European Union (EU) priority PAHs (required by the Recommendation 2005/108/EC) in vegetable oils. Two spectrofluorometric detectors and a UV-Visible detector in series were used to identify and quantify the target compounds. Linearity, recoveries, LOD, and LOQ were found to be in agreement with the performance criteria for benzo[a]pyrene (BaP) analysis as required by the Commission Directive 2005/10/EC, and satisfactory for all the compounds of interest, except for cyclopenta[c,d]pyrene, which presented a very low signal in the UV. Optimised chromatographic conditions for the separation of 25 PAHs, comprising both EPA and EU priority PAHs plus benzo[e]pyrene and benzo[b]chrysene, have been also proposed.     

Development and optimisation of a dopant assisted liquid chromatographic-atmospheric pressure photo ionisation-tandem mass spectrometric method for the determination of 15+1 EU priority PAHs in edible oils

European food legislation defines a set of 16 polycyclic aromatic hydrocarbons (PAHs) as of high concern for human health. The EU set contains structurally very similar PAHs with ring numbers between 4 and 6, and so raises some separation aspects and problems, which were not experienced with traditionally analysed PAHs. Many of the currently applied gas chromatographic mass spectrometric (GC-MS) methods suffer from separation problems, while high performance liquid chromatography with fluorescence detection (HPLC-FLD) is neither capable of detecting the whole set of EU priority PAHs nor does it (compared to GC-MS) allow structural identification. In addition HPLC-FLD shows limitations with difficult matrices due to interferences. The aim of this paper is to fill this gap by describing a liquid chromatographic dopant assisted atmospheric pressure photo ionisation tandem mass spectrometric (LC-DA-APPI-MS/MS) method for the determination of 15+1 EU priority PAHs in edible oil, which complies with the requirements set by European food legislation. Measurements were performed in positive ion mode. Anisole at a flow rate of 30 μl/min was used as dopant. Sample preparation was performed offline by donor-acceptor complex chromatography (DACC). Compared to HPLC-FLD methods the presented method enables the determination of all 15+1 EU priority PAHs at the low μg/kg concentration range including less fluorescence active compounds like benzo[j]fluoranthene and indeno[1,2,3-cd]pyrene. By analysing four reference materials it could be demonstrated that this method provides accurate results and is sufficiently sensitive for food control purposes. Statistically significant differences between the reference values and the measured analyte contents were not found. The method performs well also for very complex samples. Repeatability relative standard deviations (RSDr) of the determination of the target PAHs in olive oil were for most analytes below 5%. The limit of detection (LOD) of the method met the requirement set by EU legislation (0.3 μg/kg).     

Composition and analysis of liquid smoke flavouring primary products

Smoke flavourings are produced on a large scale and have been applied to a variety of food products for more than 30 years. The use of them has many advantages compared to traditional smoking techniques. Among others, the amount of (known) toxic compounds deriving from combustion processes can be more easily controlled in smoke flavourings. In order to ensure safe products, a new European Regulation requests data on the composition and lays down, in particular, the maximum permitted concentrations for selected polycyclic aromatic hydrocarbons (PAHs). This review compiles results published on the chemical composition of liquid smoke flavouring primary products, partly in relation to production process parameters, and the analytical methods involved. The methods cover chromatographic techniques for analysis of specific compounds including extraction methods and clean-up procedures. Analysis of sensorial and bulk parameters such as acidity and total phenolic compounds are described as well as they are used as standard methods for analysis of liquid smoke flavourings. A special section is devoted to discussing the analysis of PAHs.     

Selective immunoclean-up followed by liquid or gas chromatography for the monitoring of polycyclic aromatic hydrocarbons in urban waste water and sewage sludges used for soil amendment

A selective clean-up procedure using immunoaffinity solid-phase extraction was applied for the trace-level determination of polycyclic aromatic hydrocarbons (PAHs) in urban waste water and sewage sludges used for soil amendment. Anti-pyrene antibodies have been immobilized on a silica-based sorbent and the cross-reactivity of the antibodies towards structurally related compounds were allowed to extract the whole class of priority PAHs. The selectivity of the antibodies provided clean extracts from sludges and, therefore, the identification and quantification were shown to be easier using either liquid chromatography (LC) with UV diode array and fluorescence detection in series or gas chromatography-mass spectrometry (GC-MS), although some loss of up to 50% was observed for the clean-up. The identification of the PAHs by matching of UV and MS spectra was greatly improved. The procedure, including immunoclean-up and LC coupled to diode array and fluorescence detection, was validated using certified reference materials with native PAHs of concentrations in the range of 0.57-2.16 mg/kg (dry sludges).     

Use of ionic liquid aggregates of 1-hexadecyl-3-butyl imidazolium bromide in a focused-microwave assisted extraction method followed by high-performance liquid chromatography with ultraviolet and fluorescence detection to determine the 15+1 EU priority PAHs in toasted cereals ("gofios")

A focused-microwave assisted extraction method using aggregates of the ionic liquid (IL) 1-hexadecyl-3-butylimidazolium bromide (HDBIm-Br) followed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and single-channel fluorescence detection (FLD) has been developed for the determination of polycyclic aromatic hydrocarbons (PAHs) in toasted cereals (“gofios”) of different nature (wheat, barley, rye, and maize corn) from the Canary Islands, Spain. The optimized HPLC-UV-vis/single-channel FLD method takes 40 min for the chromatographic run with limits of detection varying between 0.02 and 4.01 ng mL⁻¹ for the fluorescent PAHs from the European Union (EU) priority list in foods, and 20.5 ng mL⁻¹ for the non-fluorescent PAH cyclopenta[c,d]pyrene (CPP). The optimized microwave step presented extractions recoveries ranging from 70.1 to 109% and precision values lower than 12.6% (as relative standard deviation), using an extraction time of 14 min. The extraction method also utilizes low amounts of sample (0.1 g), and low amounts of IL (77 mg), avoiding completely the use of organic solvents.     

Determination of Benzo[a]pyrene in smoking-flavour agents (water-soluble liquid smoke) by second derivative constant-wavelength synchronous spectrofluorimetry

We have developed a simple, rapid, inexpensive method for the determination of benzo[a]pyrene (BP, a known carcinogen) in smoking-flavour agents (water-soluble liquid smoke; WSLS). After purification of the WSLS by a single passage through a Sep Pak C18 Plus cartridge, BP in the hexane eluate was determined by second derivative constant-wavelength synchronous spectrofluorimetry. Method precision (RSD < 6%) and recovery ( approximately 92%) were satisfactory, and the detection and quantification limits (1.05 and 2.28 mug kg(-1) respectively) indicated that the current maximum permissible concentration of BP in smoke flavourings (10 mug kg(-1)) can be monitored by this method.     

In vivo quantification and pharmacokinetic studies of cotinine in mice after smoke exposure by LC–MS/MS

A sensitive analytical method was developed and validated for the quantification of cotinine in mouse plasma after exposure to smoke of 0.5, 1.0, and 1.5 commercially available cigarettes, using liquid chromatography tandem mass spectrometry. The method was validated over a linear concentration range of 0.075–20.0 ng/mL with the R² value being higher than 0.99. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. The lower limit of quantification (LLOQ) for cotinine was 0.075 ng/mL with sufficient specificity, accuracy, and precision. Following exposure to 0.5, 1.0, and 1.5 cigarette smoke, it was observed that the AUC and the C_max increased linearly as the doses increased. The pharmacokinetics of cotinine was found linear for the range of 0.5–1.5 commercial cigarette smoke. The quantification of the concentration of cotinine in mouse plasma after smoke exposure will facilitate future behavioral and toxicological experiments in animals and may prove useful in predicting cotinine levels in humans during smoking.     

Development and validation of a liquid chromatography/atmospheric pressure photoionization-tandem mass spectrometric method for the analysis of mycotoxins subjected to commission regulation (EC) No. 1881/2006 In cereals

A sensitive and reliable liquid chromatography/photoionization (APPI) tandem mass spectrometry method has been developed for determining nine selected mycotoxins in wheat and maize samples. The analytes were chosen on the basis of the mycotoxins under EU Commission Regulation (EC) No. 1881/2006, i.e., deoxynivalenol (DON), zearalenone (ZON), aflatoxins (AFs), and ochratoxin A (OTA), and considering the possibility of a near future regulation for T-2 and HT-2 toxins. Mycotoxins were extracted from samples by means of an one-step solvent extraction without any cleanup. The developed multi-mycotoxin method permits simultaneous, simple, and rapid determination of several co-existing toxins separated in a single chromatographic run, in which AFs, T-2 and HT-2 toxin are acquired in positive, while OTA, DON and ZON in negative mode. Although a moderate signal suppression was noticeable, matrix effect did not give significant differences at p = 0.05. Then, calibration in standard solution were used for quantitation. Based on the EU Commission Decision 2002/657/EC, the method was in-house validated in terms of ruggedness, specificity, linearity, trueness, within-laboratory reproducibility, decision limit (CCα) and detection capability (CCβ). For all the analytes, the regression coefficient r ranged between 0.8752 (DON in wheat) and 0.9465 (ZON in maize), biases related to mean concentrations were from −13% to +12% of the nominal spiking level, and the overall within-laboratory reproducibility ranged 3–16%; finally, CCα values did not differ more than 20% and CCβ not more than 42% from their respective maximum limit. Method quantification limits ranged from 1/20 (AFG1) to 1/4 (AFG2 and OTA) the maximum limit established by European Union in the Commission Regulation (EC) No. 1881/2006 and its subsequent amendments.     

Micellar microwave-assisted extraction combined with solid-phase microextraction for the determination of polycyclic aromatic hydrocarbons in a certified marine sediment

A method for the determination of polycyclic aromatic hydrocarbons (PAHs) in marine sediments using microwave-assisted extraction with a micellar medium combined with solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) has been developed. Two kinds of SPME fibers (100 μm polydimethylsiloxane and 85 μm polyacrylate) and different micellar media were compared for the extraction efficiency of the 16 EPA priority PAHs. The polyacrylate fiber with a micellar medium of polyoxyethylene-10-laurylether provides the highest extraction efficiency. The method is remarkable for presenting lower equilibrium times and considerably higher reproducibilities than the obtained in aqueous medium. The LODs obtained ranged between 0.28 ng/ml for fluorene and 7.66 ng/ml for indeno[1,2,3-cd]pyrene. The method has been applied to the determination of PAHs in a certified marine sediment (SRM 1941a), obtaining recoveries between 58.6 and 111.5% for three- to five-ring PAHs with precision close to or lower than the certified values.     

Monitoring of Polycyclic Aromatic Hydrocarbon Levels in Mussels (Mytilus galloprovincialis) from Aquaculture Farms in Central Macedonia Region,Greece, Using Gas Chromatography–Tandem Mass Spectrometry Method

A new sensitive and selective gas chromatography tandem mass spectrometry (GC-MS/MS) method was developed for the analysis of 26 polycyclic aromatic hydrocarbons (PAHs), including 16 Environmental Protection Agency (EPA) and 15 + 1 European Union (EU) PAHs, in mussel samples from aquaculture farms in Thermaikos and Strymonian Gulf, Central Macedonia Region, in three sampling periods. Concentrations were found at moderate to low values at all sampling sites, without exceeding maximum levels set by EU. Low molecular weight PAHs were predominant in all samples. Seasonal variation of the concentrations was observed; values were slightly higher in the winter period. Use of diagnostic ratios for potential sources of PAHs showed both petrogenic and pyrolitic origin. In comparison to other related studies of mussels from the Mediterranean Sea, Greek mussels cultivated in the studied gulfs are low in contaminants due to minimal environmental pollution effects. Low concentrations of PAHs are in compliance with the low values of other POPs which were found in the mussels.     

Optimisation of the GC-MS conditions for the determination of the 15 EU foodstuff priority polycyclic aromatic hydrocarbons

European legislation has recently established a list of 15 priority polycyclic aromatic hydrocarbons (PAHs) to be monitored in foodstuff. Thus, the accurate determination of these compounds has become a highly relevant issue. The fact that some of these European Union (EU) PAHs differ from those typically analysed, requires the re-evaluation of the instrumental conditions for the proper determination of the new target compounds. In this study, the influence of the stationary phase and dimensions of the GC capillary column on the chromatographic resolution of the 15 EU PAHs has been investigated. Apolar (DB-5 type) and medium polar (DB-17 type) stationary phases with different lengths and film thickness have been evaluated for the separation of the target compounds, with special emphasis on those coelutions involving isomers such as the three benzofluoranthenes included in the EU PAHs. In addition, the influence of the injection technique and the column dimensions on the recovery of the high molecular mass PAHs has been studied. A programmable temperature vaporising (PTV) injector has been used in three different operational modes and the results were compared to those obtained using on-column injection. The experimental parameters involved in the injection step were optimised by using experimental design.     

Low-pressure gas chromatography-ion trap mass spectrometry for the fast determination of polycyclic aromatic hydrocarbons in air samples

A low-pressure gas chromatography-ion trap mass spectrometry (LPGC-ITMS) method was investigated to shorten the analysis time for 18 US Environmental Protection Agency priority listed polycyclic aromatic hydrocarbons (PAHs). Their elution was optimised with a short, wide-bore column coupled to a deactivated capillary at the inlet end and with a long, conventional column to compare their analytical performance. The analytical figures of merit under optimal LPGC-ITMS conditions were determined with respect to chromatographic separation, S/N ratio, limit of detection and precision. The peak width at half height of 1.5s matched the ITMS duty cycle. Up to 16 PAHs in the molecular weight (MW) range of 128-278 Da could be separated in a very short time, i.e. less than 13 min using LPGC-ITMS, whereas with conventional GC-MS, it took approximately 40 min. However, LPGC-ITMS has a limited loss of separation power compared to that of conventional GC-MS due to the occurrence of three critical pairs for high-MW PAHs. For a practical evaluation, the LPGC-ITMS approach was applied to the determination of PAHs in gas and aerosol phase samples collected in the ambient air of Hasselt, Belgium.     

ASE-SPE/GC-MS测定土壤中16种PAHs质量控制研究

优化了土壤中16种优控多环芳烃( PAHs)的分析方法,建立了一套完备的质量控制体系,解决了PAHs分析中常见的技术难点,如苯并(a)芘(BaP)回收率低,基质复杂的样品净化效果不理想,萘(Nap)和菲(Phe)挥发损失和环境本底影响等.样品经加速溶剂提取(ASE),固相萃取(SPE)净化,逐级减压浓缩,气相色谱质谱( GC - MS)测定,并以氘代苯并a芘(BaP - d12)作回收率指示物.实验比较了3种正相SPE吸附剂的效果,发现弗罗里硅土对BaP存在明显的降解现象,BaP的定量应使用同位素稀释法,以降低其分析不确定度;氧化铝对PAHs的吸附性过强,不利于样品净化;硅胶最为理想.PAHs的仪器检出限为0.26～5.7 pg,方法检出限为0.067 ～0.97 ng/g(干重),土壤基质加标回收率为71％～ 122％,相对标准偏差为1.6％～8.3％.将该法用于7个电子废物焚烧区域农田土壤样品的测定,PAHs含量在28～ 283 ng/g(干重)之间,样品中BaP-d12的回收率为90％～124％,各项质控指标符合检测要求.     

Development and validation of a novel RP-HPLC method for the analysis of reduced glutathione

The objective of this study was the development, optimization, and validation of a novel reverse-phase high-pressure liquid chromatography (RP-HPLC) method for the quantification of reduced glutathione in pharmaceutical formulations utilizing simple UV detection. The separation utilized a C18 column at room temperature and UV absorption was measured at 215 nm. The mobile phase was an isocratic flow of a 50/50 (v/v) mixture of water (pH 7.0) and acetonitrile flowing at 1.0 mL/min. Validation of the method assessed the methods ability in seven categories: linearity, range, limit of detection, limit of quantification, accuracy, precision, and selectivity. Analysis of the system suitability showed acceptable levels of suitability in all categories. Likewise, the method displayed an acceptable degree of linearity (r(2) = 0.9994) over a concentration range of 2.5-60 μg/mL. The detection limit and quantification limit were 0.6 and 1.8 μg/mL respectively. The percent recovery of the method was 98.80-100.79%. Following validation the method was employed in the determination of glutathione in pharmaceutical formulations in the form of a conjugate and a nanoparticle. The proposed method offers a simple, accurate, and inexpensive way to quantify reduced glutathione.     

Trace-level analysis of polychlorinated biphenyls,organochlorine pesticides and polycyclic aromatic hydrocarbons in human plasma or serum by dispersive liquid–liquid microextraction and gas chromatography–tandem mass spectrometry

A method to determine 8 polychlorinated biphenyls (PCBs), 23 organochlorine pesticides (OCPs) and 16 polycyclic aromatic hydrocarbons (PAHs) was described using dispersive liquid–liquid microextraction (DLLME) of a small amount of plasma or serum sample and gas chromatography–tandem mass spectrometry (GC–MS/MS). The appropriate selection of the extraction solvent and dispersing solvent contributes to a high extraction yield and a clean extract. To verify the developed method, the interference, linearity of the calibration curve, detection limit, precision and accuracy were evaluated. The calibration curves were linear by 2–3 orders of magnitude with correlation coefficients above 0.997 in all cases. The LODs of PCBs, OCPs and PAHs were measured in the ranges of 0.0006–0.0029, 0.001–0.029 and 0.0002–0.012 ng/mL. The intraday precision achieved by this method was 2.19–10.3% (PCBs), 1.65–14.3% (OCPs) and 0.91–12.8% (PAHs), and the intraday accuracy 1.56–7.37% (PCBs), 2.34–19.6% (OCPs) and 1.49–15.7% (PAHs). The advantage of this method is that the analysis of PCBs, OCPs, and PAHs can be performed in a single chromatographic run, and the low detection limit enables monitoring of target substances in low exposure general public samples, and the analysis procedure is relatively simple and fast.     

Quantification of 13 priority polycyclic aromatic hydrocarbons in human urine by headspace solid-phase microextraction gas chromatography-isotope dilution mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants in both living and working environments. The aim of this study was the development of a headspace solid-phase microextraction gas chromatography-isotope dilution mass spectrometry (HS-SPME/GC-IDMS) method for the simultaneous quantification of 13 PAHs in urine samples. Different parameters affecting PAHs extraction by HS-SPME were considered and optimized: type/thickness of fiber coatings, extraction temperature/time, desorption temperature/time, ionic strength and sample agitation. The stability of spiked PAHs solutions and of real urine samples stored up to 90 days in containers of different materials was evaluated. In the optimized method, analytes were absorbed for 60 min at 80 °C in the sample headspace with a 100 μm polydimethylsiloxane fiber. The method is very specific, with linear range from the limit of quantification to 8.67 × 10³ ng L⁻¹, a within-run precision of <20% and a between-run precision of <20% for 2-, 3- and 4-ring compounds and of <30% for 5-ring compounds, trueness within 20% of the spiked concentration, and limit of quantification in the 2.28-2.28 × 10¹ ng L⁻¹ range. An application of the proposed method using 15 urine samples from subjects exposed to PAHs at different environmental levels is shown.     

Multifactorial optimization approach for the determination of polycyclic aromatic hydrocarbons in river sediments by gas chromatography-quadrupole ion trap selected ion storage mass spectrometry

A procedure for the determination of very low polycyclic aromatic hydrocarbons (PAHs) concentrations in sediment samples has been developed by gas chromatography-quadrupole ion trap mass spectrometry (GC-QIT MS) after extraction with dichloromethane and purification by using silica gel cleanup. Identification and quantification of analytes were based on the selected ion storage (SIS) strategy using deuterated PAHs as internal standards. In order to search out the main factors affecting the SIS mass spectrometry efficiency, four MS parameters, including target total ion count (TTIC), waveform amplitude (WA), transfer line (XLT) and ion trap temperatures (ITT) were subjected to a complete multifactorial design. The most relevant parameters obtained (TTIC and WA) were optimized by a rotatable and orthogonal composite design. Optimum values for these parameters were selected for the development of the method involving PAH determination in sediment samples. The optimized method exhibited a range of 111-760% higher signal-to-noise (S/N) ratios for PAHs in comparison with the method operated by the default conditions, demonstrating that the multifactorial optimization contributed to substantially improve the sensitivity of the GC-QIT MS determination. The accuracy of the method was verified by analyzing NWRI EC-3 certified reference material (Lake Ontario sediment). The selectivity, sensitivity (limits of quantification were in the range of 0.02-11.0 ng g(-1)), accuracy (recoveries >or=77%) and precision (RSD相似文献