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Analysis, fate and risks of organic contaminants in

2011 Sample preparation off line Y. Picó 1, M.

Barceló 2,3,4 1 SAMA Food and Environmental Safety

Environmental Chemistry, IDAEA CSIC, Barcelona, Spain 3

1 Analysis, fate and risks of organic contaminants in river basins under water scarcity Valencia, 7 8 February 2011 Sample preparation off line Y. Picó 1, M. Farré 2, D. Barceló 2,3,4 1 SAMA Food and Environmental Safety Research Group, Facultat de Farmàcia 2 Department of Environmental Chemistry, IDAEA CSIC, Barcelona, Spain 3 Catalan Institute for Water Research (ICRA), Girona, Spain 4 King Saud University, Riyadh, Saudi Arabia ANALYTICAL METHOD

EXTRACTION Liquid matrices Liquid liquid Extraction (LLE) Solid phase extraction (SPE) Microextraction

matrices Soxhlet Extraction Liquid Extraction Supercritical Fluid Extraction (SFE) Micro wave assisted

samples Liquid liquid extraction (LLE) Solvents: Ethyl acetate and dichloromethane Compounds having a

PBBs, PCBs, dioxins) Factors determining analyte recovery: Affinity of the analyte for the solvent

2 EXTRACTION Liquid matrices Liquid liquid Extraction (LLE) Solid phase extraction (SPE) Microextraction procedures o Sorptive Extraction o Liquid liquid microextraction BOTTLE NECK OF THE ANALYSIS Solid matrices Soxhlet Extraction Liquid Extraction Supercritical Fluid Extraction (SFE) Micro wave assisted extraction (MAE) Pressurized liquid extraction (PLE) Matrix Solid Phase Dispersion (MSPD) Liquid samples Liquid liquid extraction (LLE) Solvents: Ethyl acetate and dichloromethane Compounds having a broad range of polarity Hexane and petroleum ether Lipohilic compounds (e.j. PBBs, PCBs, dioxins) Factors determining analyte recovery: Affinity of the analyte for the solvent Sovent to sample volumen ratio The number of extraction steps Water ph can be adjusted to suppress analyte ionization (for basic and acid compounds)

3 Liquid samples Liquid liquid extraction (LLE) DRAWBACKS Labor intensive Time consuming Extensive use of glassware Large volume of highly purified solvents Emulsions problems Pre treatment of liquid matrices other than water Liquid samples Solid phase extraction (SPE) 1970 s Extraction and preconcentration of organic compounds in liquid matrices by passing the sample through a short column filled with an adsorbing medium followed by desorption with small quantity of an organic solvent. Easily incorporate in automated procedures Easily incorporate in on line systems Advantages over LLE: Faster More reproducibe Emulsions are avoided Smaller sample size

4 Solid phases Abbreviation C 18 SDVD GCB Envichrom P Octadecyl silice Highly cross linked styrene divinylbenzene or PRP 1 Graphitized Carbon Black (Carboparck, Carbograph or Envicarb) Styrene/divinyl benzene co polymer Lichrolut Reverse phase C 18 Oasis HLB Hydrophilic Lipophilic Balance (poli (divinilbenzenoco N vinilpirrolidona) Liquid samples Solid phase extraction (SPE)

Liquid samples Solid phase extraction (SPE) Vazquez Roig, P.; Blasco, C.

SPE and LC MS/MS determination of 14 illicit drugs in surface waters from the

Analytical and Bioanalytical Chemistry.397,pp. 2851 2864, 2010.

observed among the several trademarks of the same type of cartridges

5 Liquid samples Solid phase extraction (SPE) Vazquez Roig, P.; Blasco, C.; Andreu, V.; Picó, Y.SPE and LC MS/MS determination of 14 illicit drugs in surface waters from the Natural Park of L'Albufera (València, Spain).Analytical and Bioanalytical Chemistry.397,pp , Selected cartridges with 60 mg of sorbents were tested with 50 ml of spiked water (absolute recoveries are shown). No difference in the extraction performance for the studied compounds was observed among the several trademarks of the same type of cartridges Comparison of the absolute recovery percentage for the illicit drugs obtained by SPE with Oasis HLB, Oasis Wax, Supelselect HLB, Strata X and Strata XCW(matriz: L Albufera lake water spiking level 50 ng/l and size of the cartridges 60 mg/3 ml)

from L Albufera and spiking level 50 ng/l).

6 OASIS HLB OASIS MCX Influence of ph adjustment, (ph 3, ph 5 and ph 7), on the absolute recoveries (matrix surface water from L Albufera and spiking level 50 ng/l). SELECTIVE SOLID PHASE EXTRACTION Immunoaffinity columns Molecularly imprinted polymers

7 Liquid samples MICROEXTRACTION PROCEDURES Liquid samples Sorptive Extraction Solid phase microextraction (SPME) Pawliszyn et al ml ID uncoated or coated fiber

phase microextraction (HS SPME) SPME HS SPME HS SPME: When placed into the headspace gas volatile

8 Liquid samples Solid phase microextraction (SPME) (a) SPME Extraction Holder SPME (b) Thermal desorption (c) HPLC Interface Desorption chamber Waste To the column Mobil phase Head Space Solid phase microextraction (HS SPME) SPME HS SPME HS SPME: When placed into the headspace gas volatile compounds adsorb onto the fibre Gas phase Volatile analytes Liquid phase Sample, dilution solvent and matrix modifier

9 In tube Solid phase microextraction (in tube SPME) Modes of in tube extraction:(a) passive (b) dynamic Different implementations of in tube SPME Liquid samples Stir Bar sorptive extraction (SBSE)

Liquid samples Stir bar sorptive extraction

chromatography mass spectrometry applied to

10 Liquid samples Stir bar sorptive extraction (SPME) Desorption in a solvent SBSE (Stir Bar Sorptive Extraction) LC (Liquid Chromatography) Blasco, C.; Font, G.; Picó, Y.Solid phase microextraction liquid chromatography mass spectrometry applied to the analysis of insecticides in honey. Food Additives and Contaminants.25,pp : SPME OF ORGANOPHOSPHORUS PESTICIDES IN HONEY Recovery % of the organophosphorus insecticides by each fiber. Conditions for the SPME were optimized in honey samples spiked at 0.1 µg/g of each studied pesticides and desorbed off line with 1 ml of methanol stirring for 15 min.

Blasco, C.; Font, G.; Picó, Y.Solid phase microextraction liquid chromatography mass spectrometry applied to the analysis of insecticides in honey. Food Additives and Contaminants.25,pp. 56 66: 2008.

. Conditions for the SPME were optimized in honey samples spiked at 0.1 µg/g of each studied pesticides and desorbed off line with 1 ml of methanol stirring for 15 min. 0.5 g 1 g 1.

11 Blasco, C.; Font, G.; Picó, Y.Solid phase microextraction liquid chromatography mass spectrometry applied to the analysis of insecticides in honey. Food Additives and Contaminants.25,pp : Recovery % SPME OF ORGANOPHOSPHORUS PESTICIDES IN HONEY Recovery % of the organophosphorus insecticides with different amount of honey using the PDMS/DVB fiber.. Conditions for the SPME were optimized in honey samples spiked at 0.1 µg/g of each studied pesticides and desorbed off line with 1 ml of methanol stirring for 15 min. 0.5 g 1 g 1.5 g Blasco, C.; Font, G.; Picó, Y.Solid phase microextraction liquid chromatography mass spectrometry applied to the analysis of insecticides in honey. Food Additives and Contaminants.25,pp : SPME OF ORGANOPHOSPHORUS PESTICIDES IN HONEY Peak areas with the extraction time using 0.5 g of sample and the PDMS/DVB fiber Conditions for the SPME were optimized in honey samples spiked at 0.1 µg/g of each studied pesticides and desorbed off line with 1 ml of methanol stirring for 15 min.

Liquid samples Liquid Liquid Microextraction (LLME) Single drop and liquid phase microextraction techniques: A direct immersion SDME (twophase), B direct immersion SDME (three phase), C headspace

Liquid samples SDME SDME is based on the suspension of a single droplet of organic solvent from the end of a microsyringe needle in an aqueous solution (Fig.

12 Liquid samples Liquid Liquid Microextraction (LLME) Single drop and liquid phase microextraction techniques: A direct immersion SDME (twophase), B direct immersion SDME (three phase), C headspace SDME, D U shaped HF LPME (three phase), E U shaped HF LPME (two phase), F rod shaped HF LPME (two phase). Liquid samples SDME SDME is based on the suspension of a single droplet of organic solvent from the end of a microsyringe needle in an aqueous solution (Fig. A) A droplet of organic solvent was suspended at the needle tip in a stirred aqueous sample (Fig A) The organic droplet held above the aqueous sample solution is most suitable for examination of volatile or semivolatile analytes (Fig. C) In this method, the analytes are first extracted from an aqueous sample matrix (donor phase) into an organic solvent (organic membrane) layered over the donor phase and are then backextracted into an aqueous drop (acceptor phase) suspended from the tip of a microsyringe into the organic membrane (Fig. B) Single drop and liquid phase microextraction techniques: A direct immersion SDME (twophase), B direct immersion SDME (three phase), C headspace SDME

Liquid samples Mebrane assisted Microextraction Analytes are extracted into the intermediary organic phase (represented by the

D) Another mode of HF LPME is based on a two phase system in which the organic solvent is used to fill both the wall pores and the

rodlike configuration (Fig. F) D U shaped HF LPME (three phase), E U shaped HF LPME (two phase), F rod shaped HF LPME (two phase).

13 Liquid samples Mebrane assisted Microextraction Analytes are extracted into the intermediary organic phase (represented by the supported liquid membrane) and then subsequently into the aqueous phase (Fig. D) Another mode of HF LPME is based on a two phase system in which the organic solvent is used to fill both the wall pores and the HF lumen (Fig E) In addition to these U shaped configurations, HF LPME has recently attracted attention for in vial extraction in a rodlike configuration (Fig. F) D U shaped HF LPME (three phase), E U shaped HF LPME (two phase), F rod shaped HF LPME (two phase). Solid samples Soxhlet extraction Condenser Extraction chamber Boiling flask Drawbacks Inavility to provide agitation The constant heat applied High volume of organic solvents Long procedure

Soxhlet Method Apolar analytes (PAHs, organochlorine, etc) Sample Dried

solid sample with a suitable solvent Blending systems Manually shaking High

Acetonitrile Methanol Ethyl acetate with anhydrous sodium sulfate 30 % of

14 Soxhlet Method Apolar analytes (PAHs, organochlorine, etc) Sample Dried Grinding Solid samples Liquid phase extraction Blending a finely dispersed solid sample with a suitable solvent Blending systems Manually shaking High speed homogenizer machines Ultrasonication bath Extractants Acetone Acetonitrile Methanol Ethyl acetate with anhydrous sodium sulfate 30 % of procedures to analyze veterinary drugs in food 80 % of procedures to determine pesticides in fruit and vegetables

Solid samples QuEChERS AOAC Approach Figure 1 Figure 2 Figure 3 Figure 4 Sample Extraction

product (Figure 1). 2. Weigh 15g of homogenized product into a clean 50mL tube (cat.

Add 15mL of 1% acetic acid in acetonitrile (v/v) and an appropriate amount of an internal

# 26214) to each extracted sample (Table II, Figure 3). 4.

Centrifuge for 1 minute at >1,500 rcf to separate the solid material (Figure 5).

Figure 5 Solid samples QuEChERS AOAC Approach Figure 6A Figure 6B Figure 7 Figure 9 For

graphitized carbon per ml For samples with colored extracts containing greater than 1%

all other samples: Clean up with 50mg PSA and 150mg MgSO4 per ml 1.

15 Solid samples QuEChERS AOAC Approach Figure 1 Figure 2 Figure 3 Figure 4 Sample Extraction 1. Homogenize the frozen commodity to generate a uniform sample representative of the product (Figure 1). 2. Weigh 15g of homogenized product into a clean 50mL tube (cat.# 26227) (Figure 2). 3. Add 15mL of 1% acetic acid in acetonitrile (v/v) and an appropriate amount of an internal standard solution. Add the contents of a Resprep Q150 tube (cat.# 26214) to each extracted sample (Table II, Figure 3). 4. Shake vigorously for 1 minute by hand (Figure 4). 5. Centrifuge for 1 minute at >1,500 rcf to separate the solid material (Figure 5). Proceed with sample clean up. Figure 5 Solid samples QuEChERS AOAC Approach Figure 6A Figure 6B Figure 7 Figure 9 For samples with greater than 1% fat: Clean up with 50mg PSA, 150mg MgSO4, and 50mg C18 per ml of extract. For samples with colored extracts: Clean up with 50mg PSA, 150mg MgSO4, and 50mg graphitized carbon per ml of extract. For samples with colored extracts containing greater than 1% fat: Clean up with 50mg PSA, 150mg MgSO4, 50mg C18, and 50mg graphitized carbon per ml of extract. For all other samples: Clean up with 50mg PSA and 150mg MgSO4 per ml of extract. 1. Using the centrifuged sample extracts, transfer the supernatant to the dspe tube as shown in Figures 6A & 6B. 2. Shake vigorously for 30 seconds (Figure 7). 3. Centrifuge for 1 minute at >1,500 rcf to separate thesolid material (Figure 8). 4. Transfer sample to an autosampler vial and test using GC or LC methods. Additional steps to prepare the sample for specific types of analysis are addressed in AOAC Official Method

Solid samples Supercritical Fluid Extraction (SFE) On an industrial scale, SFE has been applied for many years and only recently has been deployed in the analytical field for extracting a variety

16 Solid samples Supercritical Fluid Extraction (SFE) On an industrial scale, SFE has been applied for many years and only recently has been deployed in the analytical field for extracting a variety toxicants of pesticides from solid matrices, such as soil, sediments, vegetables, animal tissues and foodstuffs. Carbon dioxide has been by far the most used fluid. Besides offering advantages mentioned below, this fluid has a low critical temperature (31 C). Advantages Non toxic, non inflamable CO2 is used as solvent SFE can be automated and coupled to both GC and LC Selective extraction modifying the density of the supercritical fluid (varying pressure and temperature) Fast extraction. Solid samples Microwave assisted extraction (MAE) refrigerant MICROWAVE OVEN microwave irradation siphon distillation flask controller heat source The microwave part of the electromagnetic spectrum is defined as energy of wavelength between mm corresponding to a frequency range of GHz. MAE is the process that use this energy to heat solid sample solvent mixture resulting in the partitioning of the compounds of interest from the solid matrix into the solvent.

Solid samples Pressurized Liquid Extraction (PLE) PLE extraction can be

continuously through the device, or in a combination of both modes.

automation and many samples can be extracted sequentially.

Extraction Water 110 ºC 1500 psi 2 min 3 cycles,7 min,100 flush % 2 g of fish

s HOMOGENIZE Put in 22 ml stain steal cell Washing 4 ml dof ammonium acetate

40 ml Oasis WAX Cartridges of 3cc Preconditioning 4 ml de 0.

17 Solid samples Pressurized Liquid Extraction (PLE) PLE extraction can be performed in a static mode, a dynamic mode, in this case the solvent flows continuously through the device, or in a combination of both modes. A very interesting feature of this technique is the possibility of full automation and many samples can be extracted sequentially. Extraction procedures: Extractant Temperature Pressure Preheating time Extraction Water 110 ºC 1500 psi 2 min 3 cycles,7 min,100 flush % 2 g of fish Ca. 25 g of sea sand 100 µl I.S.s HOMOGENIZE Put in 22 ml stain steal cell Washing 4 ml dof ammonium acetate buffer 25 mm (ph 4) 4 ml of methanol Clean-up the aqueous extract Ca. 40 ml Oasis WAX Cartridges of 3cc Preconditioning 4 ml de 0.1 % NH4OH in methanol 4 ml methanol 4 ml water Elution 4 ml 0.1 M NH4OH in methanol Evaporate at 0.5 ml and inject in the LC- MS M. Llorca, M. Farré, Y. Picó, D. Barceló, J. Chromatogr. A 43 (2009)

Solid samples Matrix Solid Phase Dispersion C-18 ADVANTAGES Analytical protocol

eliminated; Solvent consumption is substantially reduced; Extraction efficiency

that it can be used for extracting analytes from both solid and liquid foods.

18 Solid samples Matrix Solid Phase Dispersion C-18 ADVANTAGES Analytical protocol is drastically simplified and shortened Possibility of emulsion formation is eliminated; Solvent consumption is substantially reduced; Extraction efficiency of the analytes is enhanced An interesting feature of the MSPD technique is that it can be used for extracting analytes from both solid and liquid foods. DETERMINATION OF ORGANOPHOSPORUS INSECTICIDES IN HONEY BY DIFFERENT EXTRACTION TECHNIQUES

19 CCα and CC β obtained for the selected pesticides in honey by SPME, PLE, QuEChERS and SPE with LC-MS 2 Pesticides SPME PLE QuEChERS SPE CCα CC β CCα CC β CCα CC β CCα CC β Fenoxycarb Phenthoate FonoFos Diazinon Phosalone Pyrazophos Chlorpyrifos methyl Profenofos Pirimiphos ethyl Temephos Bromophos ethyl Chlorpyrifos ethyl Techniques of sample extraction and purification ++ main application, + secondary application, -generally no application. Technique Extraction Purificati Characteristics on LSE ++ Laborious, highly time consuming, large solvent volume, purification step needed Soxhlet extraction ++ Laborious, highly time consuming, large solvent volume, no filtration required MAE ++ Purification step needed, low solvent volume, but solvent must be able to absorb microwaves, evaporated temperature ASE ++ Purification step needed, low solvent volume, evaporated temperature SPE ++ + Easily automated, low solvent volumes SPME ++ Decreased or no use of solvents, easily automated, no sensitivity to suspended matter, problems with reproducibility and optimization MSPD ++ + Low solvent volume, not very suitable for dry samples or samples with high lipid content adsorbent consumption is then relatively high and MSPD requires an additional clean up step GPC ++ Easily automated, long lifespan of columns, good recovery, highly time consuming Column chromatography ++ Laborious, highly time consuming Liquid chromatography ++ Easily automated, low solvent volumes

20 Thank you very much for your attention! Questions

What type of samples are common? Time spent on different operations during LC analyses. Number of samples? Aims. Sources of error. Sample preparation

What type of samples are common? Sample preparation 1 2 Number of samples? Time spent on different operations during LC analyses 3 4 Sources of error Aims Sample has to be representative Sample has to