Agilent Analysis of Polycyclic Aromatic Hydrocarbons in Fish with Agilent Bond Elut QuEChERS AOAC Kit HPLC-FLD Manual
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1. e e e J Authors Bellah 0 Pule Lesego C Mmualefe Nelson Torto Department of Chemistry Rhodes University P 0 Box 94 Grahamstown 6140 South Africa Analysis of Polycyclic Aromatic Hydrocarbons in Fish with Agilent Bond Elut QuEChERS AOAC Kit and HPLC FLD Application Note Food Abstract An HPLC Florescence Detection FLD method was developed and validated for the determination of sixteen polycyclic aromatic hydrocarbons PAHs in fish fillets The analyzed compounds included naphthalene Nap acenaphthylene Acy acenaph thene Ace fluorene Flu phenanthrene Phe anthracene Ant fluoranthene Fin pyrene Pyr 1 2 benza aJanthracene BaA chrysene Chr benzo e pyrene BeP benzo e acenaphthylene BeA benzo k fluoranthene BkF dibenzo a hJanthracene DahA benzo g h i perylene Bghi P and indeno 1 2 3 cd pyrene InP The method employs a quick easy cheap effective rugged and safe QUEChERS multiresidue sample preparation procedure adopted from the Association of Analytical Communities AOAC Official method 2007 01 for extraction and cleanup The analytes were separated on an Agilent ZORBAX Eclipse PAH HPLC column 4 6 mm x 50 mm 1 8 um by gradient elution with a binary system of acetonitrile water and subse quent fluorescence detection set at appropriate excitation and emission wavelengths The analyte recoveries ranged from 83 4 to 101 with relative standard deviations ranging from 02. of the upper CH CN layer was transferred into a Bond Elut QUEChERS AOAC Dispersive SPE 15 mL tube This SPE tube contained 400 mg of PSA 400 mg of C18EC and 1200 mg of anhydrous MgSO After one minute of shak ing the tubes were centrifuged at 4000 rpm for 5 min A 4 mL aliquot of the extract was filtered through a 0 45 um PVDF syringe filter then 1000 uL of the extract was placed in an autosampler vial for HPLC FLD analysis Weigh 5 g homogenized fish sample into a 50 mL centrifuge tube Y Spike samples with 2000 uL spiking solution Shake vigorously 1 min Add 8 mL ACN Shake vigorously 1 min Add Bond Elut QUEChERS AOAC salt packet Shake 1 min centrifuge at 4000 rpm 5 min Transfer 6 mL aliquot to Bond Elut QUEChERS Dispersive SPE 15 mL tube Shake 1 min centrifuge at 4000 rpm 5 min Filter through a 0 45 um PVDF syringe filter i Transfer 1 mL extract to an autosampler vial Y Samples are ready for HPLC FLD analysis Figure 2 Flow chart of QUEChERS AOAC sample preparation procedure Results and Discussion Chromatographic results Figure 3 shows an overlay of color coded chromatograms at various fluorescence conditions Table 1 of the standard mix ture of the 16 PAHs A chromatogram of the blank fish extract is presented in Figure 4 Overlay chromatograms of the spiked fish sample at spiking level 1 are shown in Figure 5 QuEChERS extraction
3. 6 to 1 9 at three different fortification levels The limits of detection and quantification ranged from 0 04 to 0 84 and 0 1 to 2 80 ng g respectively j Agilent Technologies Introduction Polycyclic aromatic hydrocarbons PAHs are a large group of organic compounds included in the European Union and US Environmental Protection Agency US EPA priority pollutant list because of their mutagenic and carcinogenic properties 1 Excluding smokers and occupationally vulnerable popula tions most individuals are exposed to PAHs predominantly from dietary sources 2 In the marine environment PAHs are bioavailable to marine species via the food chain as water borne compounds and contaminated sediments As lipophilic compounds they can easily cross lipid membranes and have the potential to bioaccumulate in aquatic organisms Although for most people fish and seafood represents only a small part of the total diet the contribution of this food group to the daily intake of PAHs in some individuals may be com paratively important 3 The AOAC QuEChERS method has been widely applied in the analysis of pesticides in food since it was introduced by USDA scientists 4 5 In general there are two major steps extraction and dispersive SPE cleanup The method uses a single step buffered acetonitrile extraction while simultane ously salting out water from the aqueous sample using anhy drous magnesium sulfate MgSO to induce liquid liquid pa
4. 8 9 0 8 Dibenzo a h anthracene 88 2 0 9 97 3 1 1 97 1 0 6 Benzo g h i perylene 98 4 0 8 95 5 1 6 98 2 0 7 Indeno 1 2 3 cd pyrene 91 5 1 5 97 9 0 9 94 3 0 7 UV detection at 230 nm References 1 M J Ramalhosa P Paiga S Morais C Delerue Matos M B P P Oliveira Analysis of Polycyclic Aromatic Hydrocarbons in Fish Evaluation of a Quick Easy Cheap Effective Rugged and Safe Extraction Method J Sep Sci 2009 32 3529 3538 L R Bordajandi M Dabrio F Ulberth H Emons Optimisation of the GC MS Conditions for the Determination of IS EU Foodstuff Priority Polycyclic Aromatic Hydrocarbons J Sep Sci 2008 31 1769 1778 J L Domingo Health Risks Arising From the Dietary Intake of Chemical Contaminants a Case Study of the Consumption of Edible Marine Species in Catalonia Contrbutions to Science 3 4 459 468 2007 L Zhao J Stevens Analysis of Pesticides Residues in Spinach Using Agilent Bond Elut QUEChERS AOAC kits by LC MS MS Agilent Technologies publication 5990 4248EN M Anastassiades S Lehotay Fast and Easy Multiresidue Method Employment Acetonitrile Extraction Partitioning and Dispersive Solid Phase Extraction for the Determination of Pesticide Residues in Produce 2003 86 412 431 J W Henderson Jr W Biazzo W Long Polycyclic Aromatic Hydrocarbons PAH Separation Using ZORBAX Eclipse PAH Columns Analyses from Si
5. D Loo Naphthalene Y 0 0222x 0 1366 0 9991 0 62 2 07 Acenaphthylene Y 0 0544x 0 0130 0 9993 0 25 0 83 Acenaphthene Y 0 0184 x 0 0204 0 9998 0 56 1 87 Fluorene Y 0 0323x 0 1717 0 9990 0 12 0 40 Phenanthrene Y 0 0950x 0 0086 0 9995 0 18 0 60 Anthracene Y 0 0838x 0 1265 0 9991 0 24 0 80 Fluoranthene Y 0 0247x 0 0237 0 9994 0 04 0 16 Pyrene Y 0 0218x 0 0432 0 9998 0 09 0 30 1 2 Benzanthracene Y 0 0120x 0 0103 0 9994 0 03 0 10 Chrysene Y 0 0052x 0 0086 0 9990 0 28 0 93 Benzo e pyrene Y 0 0144x 0 0037 0 9997 0 04 0 16 Benz e acenaphthylene Y 0 1186x 0 032 0 9995 0 07 0 23 Benzo k fluoranthene Y 0 0464x 0 0969 0 9997 0 05 0 16 Dibenzo a h anthracene Y 0 0531x 0 0001 0 9990 0 84 2 80 Benzofg h i perylene Y 0 0440x 0 0722 0 9993 0 11 0 36 Indeno 1 2 3 cd pyrene Y 0 0324x 0 0912 0 9993 0 05 0 18 UV detection at 230 nm Recovery and Reproducibility Table 3 PAHs Spiking Levels The recovery and reproducibility RSD were evaluated on PAH Spiking level ng g spiked samples at three different levels Table 3 The analysis 1 2 3 was performed in replicates of six n 6 at each level Table Naphthalene 20 100 200 4 shows the very good to excellent recoveries and excellent Acenaphthylene 20 100 200 RSD values for the sixteen polycyclic aromatic hydrocarbons Ac
6. The use of CH CN as an extracting solvent in a salting out condition without the need to add co solvents attained high extraction yields as shown by the recoveries in Table 4 The CH CN solvent is compatible with the HPLC FLD procedure in this application note Therefore no evaporation or reconsti tution solvent was required This is particularly important for the PAHs since some of these compounds naphthalene ace naphthene and fluorene are extremely volatile and may be lost during an evaporation step 1 Standard mixture 0 2 4 6 8 10 12 14 Time min L P Figure 3 Overlay HPLC FLD chromatograms of the standard mixture containing 1 Nap 2 Acy 3 Ace 4 Flu 5 Phe 6 Ant 7 Fin 8 Pyr 9 BaA 10 Chr 11 BeP 12 BeA 13 BkF 14 DahA 15 BghiP 16 InP The concentration of the PAHs was 1 mg mL The blue portion of the chromatogram used the following excitation emission wavelengths 260 nm 352 nm the red portion 260 nm 420 nm the light blue portion 260 nm 440 nm For acenaphthylene UV detection at 230 nm was used Chromatographic conditions are shown in Table 1 a gt 0 45 Unspiked fish extract gt 0 2 0 l 0 2 4 6 8 10 12 14 Time min No Figure 4 Chromatogram of the blank fish extract Chromatographic conditions are shown in Table 1 The baseline chromatogram used the fol lowing excitation emission wavelengths 260 nm 352 nm The other excitation emission
7. atephthene 10 50 100 7 Fluorene 10 50 100 Conclusions Phenanthrene 10 50 100 F eee Anthracene 10 50 100 A simple and fast mulitiresidue method based on Bond Elut QuEChERS AOAC and HPLC FLD has been developed for the _ orantfene ku L a simultaneous determination of sixteen polycyclic aromatic Pyrene 10 50 100 hydrocarbons at parts per billion ppb levels in fish tissue 1 2 Benzanthracene 5 20 50 High recoveries with excellent RSD were attained therefore Chrysene 10 50 100 the method should be applied for quality control of PAHs in Benzo e pyrene 5 20 50 real samples Benz e acenaphthylene 5 20 50 Benzo k fluoranthene 5 20 50 Dibenzo a h anthracene 5 20 50 Benzofg h i perylene 5 20 50 Indeno 1 2 3 cd pyrene 5 20 50 UV detection at 230 nm Table 4 Recoveries and RSDs for the Sixteen Polycyclic Aromatic Hydrocarbons in Fish Sample n 6 PAH Level of spiking ng g n 6 1 2 3 Recovery RSD Recovery RSD Recovery RSD Naphthalene 94 7 1 4 97 9 1A 93 8 1 4 Acenaphthylene 87 8 1 7 96 3 1 2 85 6 0 8 Acenaphthene 92 1 1 5 93 0 1 8 96 7 0 8 Fluorene 98 1 1 5 89 9 1 0 97 2 0 9 Phenanthrene 90 6 0 9 93 8 0 8 83 1 1 7 Anthracene 96 7 1 0 87 6 0 8 92 1 0 6 Fluoranthene 83 4 1 3 93 9 1 5 95 9 1 2 Pyrene 93 5 1 8 86 1 1 3 95 0 1 4 1 2 Benzanthracene 94 5 1 3 89 6 1 6 94 9 1 0 Chrysene 101 0 1 4 97 8 T7 87 2 1 6 Benzo e pyrene 88 8 1 5 85 2 1 9 95 0 1 4 Benz e acenaphthylene 95 5 0 7 92 7 0 7 89 2 0 9 Benzo k fluoranthene 93 5 0 8 94 6 0 9 9
8. conditions showed no other interferences O X ae Spiked fish extract Time min hs Figure 5 Overlay HPLC FLD chromatograms of the spiked fish sample containing 1 Nap 2 Acy 3 Ace 4 Flu 5 Phe 6 Ant 7 Fin 8 Pyr 9 BaA 10 Chr 11 BeP 12 BeA 13 BkF 14 DahA 15 BghiP 16 InP The spiking level for this sample was level 1 see Table 3 The blue portion of the chromatogram used the following excitation emission wavelengths 260 nm 352 nm the red portion 260 nm 420 nm the light blue portion 260 nm 440 nm For acenaphthylene UV detection at 230 nm was used Chromatographic conditions are shown in Table 1 Limits of Detection and Quantification The limits of detection and quantification were estimated from the concentration of sulfonamides required to give a signal to noise ratio of 3 and 10 respectively Table 2 shows the regression equation correlation coefficients and very acceptable limits of detection and quantification Linearity Limit of Detection LOD and Limit of Quantification LOQ Linearity The linear calibration curves were obtained by plotting the peak area for each analyte versus its concentration Curves were generated by spiking the sample blanks at a concentra tion range of 0 300 ng g Table 2 Linearity LOD and LOQ for the Sixteen Polycyclic Aromatic Hydrocarbons PAH Regression equation R2 LO
9. ene Benzo b fluoranthene Naphthene Pyrene Dibenzo a hJanthracene Fluoranthene NS QOO XR OO GE ott Phenanthrene Fluorene Indeno 1 2 3 cd pyrene Figure 1 2 Chemical structures for the polycyclic aromatic hydrocarbons used in the study Standard Solutions Standard stock solutions 1 mg mL were prepared by dis solving 10 mg of the desired PAH in 10 ml CH CN and stored at 20 C All working solutions were prepared fresh daily by serial dilution with CH CN Equipment and Material The analysis was performed on an Agilent 1200 Series HPLC Agilent Technologies Santa Clara CA USA equipped with a binary pump and a fluorescence detector FLD set at varying excitation and emission wavelengths Table 1 The selection of the excitation and emission wavelengths for detection was based on the optimum responses for the various PAHs Separation of the compounds was achieved on an Agilent ZORBAX Eclipse PAH column 4 6 mm x 50 mm 1 8 um p n 959941 918 The data was processed by HPLC 2D Chemstation software Extraction and cleanup were achieved with Agilent Bond Elut Buffered QUEChHERS AOAC Extraction kit p n 5982 5755 and Bond Elut QUEChERS AOAC Dispersive SPE kit p n 5982 5158 Agilent Technologies A Kenwood Grinder obtained from a local appliance store was employed for homogenizing the fish sample Instrument conditions HPLC conditions Table 1 HPLC Conditions used for Separa
10. r titioning After removing an aliquot from an organic layer for further cleanup a dispersive solid phase extraction dSPE step is conducted using a combination of primary secondary amine PSA sorbent to remove organic acids from other com ponents and anhydrous MgSO to reduce the remaining water in the extract Other sorbents such as graphitized carbon black GCB may be added to remove pigments and sterol or C18 to remove lipids and waxes This application note presents a method for the analysis of PAHs at trace levels in fish tissue with HPLC FLD The HPLC methods are useful for PAH analysis since UV and fluores cence detection offer enhanced selectivity over other tech niques such as GC with flame ionization detection 6 The method includes sample preparation with Bond Elut AOAC Buffered Extraction kit p n 5982 5755 and Bond Elut AOAC Fatty Dispersive SPE 15 mL kit p n 5982 5158 Chemical structures of the PAHs in this study are shown in Figure 1 Experimental Reagents and Chemicals All reagents were analytical or HPLC grade Acetonitrile CH CN and PAHs were purchased from Sigma Aldrich St Louis MO USA The water used was from a Milli system Milford Mass USA The mobile phase was filtered through a Whatman membrane filter 47 mm diameter and 2 um pore size Acenapthene Acenapthylene Anthracene Benzo k fluornthene Chrysene ococmantatots Benzo a Janthracene Dibenzo ghi perylene Benzo a pyr
11. tion of PAHs Agilent ZORBAX Eclipse PAH C18 4 6 x 50 mm 1 8 ym 0 8 mL min Column temperature 18 C Column Flow rate Injection volume 5 uL Mobile phase A Deionized H 0 B CH CN Gradient T min B 0 60 1 5 60 7 90 13 100 Detection UV at 230 nm Acy and varying fluorescence excitation Ex and emission Em wavelengths Wavelengths Time min Ex Em wavelengths nm PAH detected 0 5 dark blue 260 352 Nap Ace Flu Phe Chr 0 14 red 260 420 Ant Pyr BeP DahA BghiP 0 14 light blue 260 460 Fin 1 2 BaA BeA BkF InP Sample preparation The fish fillets were purchased from a local food store minced and deep frozen until analysis Extraction A 5 0 g sample of fish homogenate was placed into a 50 mL centrifuge tube from the Bond Elut QUEChERS AOAC Extraction kit and the tube was centrifuged for 20 s Samples were then spiked with appropriate spiking solutions to yield appropriate working solutions for recoveries and reproducibili ty studies A 2000 uL volume of spiking solution was added to all samples except the blank and the tubes were shaken vig orously for 1 min Next 8 mL of CH CN then an Agilent Bond Elut QUEChERS AOAC extraction salt packet p n 5082 5755 containing 6 g of anhydrous MgSO and 1 5 g of anhydrous NaOAc were added to the tubes The sample tubes were hand shaken vigorously for 1 min then further centrifuged at 4000 rpm for 5 min Dispersive SPE Cleanup A 6 0 mL aliquot
12. x to 24 PAHs Agilent Technologies publication 5989 7968EN For More Information For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2012 Printed in the USA January 6 2012 5990 5441EN Agilent Technologies
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