D. Cavagnino, A. Siviero, A. Mantegazza DANI Instruments, Italy V. Termopoli, P. Palma, G. Famiglini, A. Cappiello University of Urbino, Italy

Transcription

1 D. Cavagnino, A. Siviero, A. Mantegazza DANI Instruments, Italy V. Termopoli, P. Palma, G. Famiglini, A. Cappiello University of Urbino, Italy 4th Multidimensional Chromatography Workshop, Toronto, Jan 8-9, 2013

2 DANI INSTRUMENTS Foued in 1975 by a group of people from Carlo Erba Strumentazione DANI Branch Office - France DANI Instruments S.A. - Swiss DANI Branch Office - Iia DANI Instruments S.p.A. Italy (Headquarters) DANI TODAY

3 DANI INSTRUMENTS Foued in 1975 by a group of people from Carlo Erba Strumentazione DANI Branch Office - France DANI Instruments S.A. - Swiss DANI Branch Office - Iia DANI Instruments S.p.A. - Italy DANI TODAY

4 OUTLINE Backgrou a aim of the project Experimental approach Preliminary comparison GC-qMS a GC-TOFMS GC GC-TOF MS approach Conclusion OUTLINE

5 SIUD AND SIDS - INTRODUCTION Sudden Intrauterine Unexplained Death (SIUD) refers to unexpected perinatal loss as fetal death after 25 completed weeks of pregnancy Sudden Infant Death Syrome (SIDS) refers to the sudden unexplained death of infants being less than 1 year old SIDS a SIUD represent the most relevant deathcausing syromes in developed countries within the first year of life However, the true etiologic factors of those syromes are still unknown BACKGROUND

6 ENVIRONMENTAL RISK FACTORS Specific environmental risk factors as maternal smoking, alcohol a drugs abuse, are known as potential SIDS a SIUD contributors, possibly correlated to genetic alterations that uerlie such syromes Only recently also the environment pollution has been considered as an important contributor to genetic alteration In the last few years, researches on new environmental risk factors on fetal a neonatal development have been directed to the investigation of the adverse effects of eocrine disruptive compous (EDCs) In particular, prenatal exposure to organochlorine a organophosphorous pesticides (OCPs, OPPs) has been associated to critical adverse effects on fetal development BACKGROUND

7 ENVIRONMENTAL RISK FACTORS OCPs, due to their high resistance to degradation, have an ubiquitary diffusion in the ecosystem with bioaccumulation in the food chain a in humans Those contaminants pass easily from mother to fetus with transfer to placenta, cord blood, serum a maternal adipose tissues, a from mother to neonate through breastfeeding Nevertheless, information regarding the detection of these pollutants in SIDS a SIUD autopsy fiings are completely missing BACKGROUND

8 AIM OF THE PROJECT Purpose of the project is to investigate on the occurrence of those environmental pollutant residues in postmortem fetal a newborn tissues, to potentially include those compous as factor risks for sudden a unexpected death. This project is supported by fuing provided by the National Research Program a the research is carried out by the group of Prof. Cappiello of the University of Urbino in collaboration with the Lino Rossi Research Center for the Study a Prevention of Unexpected Perinatal Death a SIDS, University of Milan, Italy. Purpose of this work is evaluate different analytical approaches as GC-qMS, GC-TOFMS a GC GC-TOFMS in terms of sensitivity a results reliability BACKGROUND

9 EXPERIMENTAL APPROACH BIOLOGICAL TISSUES The extraction method was first developed a validated using swine liver a lamb brain tissues, before applying on liver a brain tissues from SIUDS a SIDS victims TARGET COMPOUNDS 20 OCPs (α-bhc, β-bhc, γ-bhc, δ-bhc, Heptachlor, Aldrin, Heptachlor epoxide, γchlordane, a-chlordane, Eosulphan I, Eosulphan II, 4,4-DDT, 4,4-DDE, 4,4DDD, Dieldrin, Erin, Erin Aldehyde, Eosulphan Sulfate, Methoxychlor, Erin ketone 2 OPPs (Chlorfenvinphos, Chlorpyrifos) 1 Phthalimide (Captan) 1 Carboxamide (Boscalid) in according to APPA (TN) (Env. Protection Agency of Trento, Italy) Bisphenol A 9 ISTD (Eosulphan I/II D4, 4,4-DDE D8, 4,4-DDT D8, Methoxychlor D14, Chlorfenvinphos D10, Chlorpyriphos D10, Captan D6, Bisphenol A D16 STANDARDS & SAMPLES

10 SAMPLE PREPARATION Solid Phase Extraction a Clean up V. C. Fernaes et al., Biomed. Chromatogr. 2012, 26, Mix 0.5g biological tissue in glass tube with 2 ml of n-hexane a IS Homogenized a taken 1 ml of supernatant Clean up: Supelco-Discovery C18, coitioned with 4 ml n-hexane, add 1 ml of extract, dry for 15 min, elute with 2 ml n-hexane a 1 ml DCM, dryness to 200 µl Animal tissues spiked with 9 isotopically labeled internal staard have been used for method validation SPE Supelco Discovery C18 with adequate homogenization offers the good performance in terms of cleanup efficiency a recoveries, with limited solvent a time consumption Good recovery has been obtained between 90% a 100% for the target analytes 1uL injected onto GC-MS EXTRACTION & CLEAN UP

11 INSTRUMENTAL METHODS GC-qMS (Agilent 5975C) GC-TOFMS (DANI MasterGC-TOF) Column HP-5MS 30m, 0.25mm id, 0.25um th Column Rxi-5ms 10m, 0.1mm id, 0.1um th Inj Vol 1uL with Agilent 7683B Autosampler Inj Vol 1uL with DANI MasterAS Autosampler Oven Initial T 80 C SSL Injector Oven Initial T 50 C SSL Injector Rate Final Temp Final Time Mode Splitless Rate Final Temp Final Time Mode Splitless 30 C/min 180 C 4.00 Temp 250 C 30 C/min 180 C 0 Temp 250 C 3 C/min 205 C 4.08 Carrier Helium 20 C/min 205 C 2 Carrier Helium 20 C/min 300 C Const Flow 1mL/min 20 C/min 300 C 2 Const Flow 0.5mL/min Run Time qms Detector Acquisition Run Time SIM TOF-MS Detector Acquisition amu The GC-TOFMS benefits have been investigated in terms of analysis time a sensitivity over the entire mass range The mass spectra information are expected to provide peaks deconvolution advantages a id confirmation at lower concentration level OPERATIVE CONDITIONS

12 DANI MASTERTOF-MS y EI Source Analyzer Internal baking pump 38 cm 51 cm z 22 cm extracted m/z 272 x Extremely compact design Hay a noise-free instrument with embedded pumping system High acquisition speed up to 1000 spectra/s Sensitivity 1 pg OFN s/n > 600:1 over the entire mass range Affordable detector for quantitative FastGC a GC GC 1pg OFN S/N RSM = pg OFN S/N RSM = 82 MASTERTOF PLUS HIGH SPEED COMPACT TIME OF FLIGHT

13 ANIMAL TISSUE SPIKED EXTRACT Instrument: DANI Master GC-TOFMS Lamb Brain Extract Sample: Extract spiked with a mixture of Column: Rxi-5ms column: 10m x 0.1mm x 0.1mm Injection volume: 1 µl splitless Mass Range: amu m/z 243 p,p DDT D8 m/z 235 p,p DDT m/z 272 Eosulfan Sulfate 1 2,

14 INSTRUMENTAL METHODS GC GC-TOFMS (DANI MasterGC GC-TOF) Modulator Zoex ZX1 Loop Modulator Columns Set 1D : DN-5MS 30m, 0.25mm id, 0.25 um th. 2D : Rxi-17 2m, 0.1mm id, 0.1um th. (0.5m loop) Modulation Time 8 secos Hot Jet Pulse C Inj Vol 1uL with DANI MasterAS Autosampler Oven Initial T 60 C SSL Injector TOFMS Detector Rate Final Temp Final Time Mode Pulsed Splitless Acquisition amu 3 C/min 310 C Temp 250 C Rate 100 Hz 94 min Carrier Helium Const Flow 0.8mL/min Run Time The GC GC-TOFMS benefits has been investigated in terms of sensitivity The improved chromatographic separation is expected to significantly reduce the detrimental matrix effect The cryogenic peak compression is also expected to work in favour of an improved s/n ratio OPERATIVE CONDITIONS

15 GC GC-TOFMS SEPARATION Staard mix in 88 ppb ( TIC profile ) 2D GC SEPARATION

16 GC GC-TOFMS SEPARATION Staard mix in 88 ppb ( TIC profile ) zoomed 2D Plot Bisphenol A 2D GC SEPARATION

17 ANIMAL LIVER TISSUE EXTRACT SPIKED 25 PPB TIC 2D PLOT 2D GC SEPARATION

18 ANIMAL LIVER TISSUE EXTRACT SPIKED 25 PPB TIC 2D PLOT Target compous fully separated from the matrix 2D GC SEPARATION

19 RIC PLOT LIVER MATRIX SPIKED AT 25 PPB

20 RIC PLOT LIVER MATRIX SPIKED AT 25 PPB

21 METHOD LINEARITY, LOD, LOQ Animal Tissue Extract spiked in the range ppb a 130 ppb α-eosulfan/p,pdde D8 2 y = x R² = Cs / Ci Cs / Ci Methoxychlor/Methoxychlor D14 40 Erin Ketone/ Methoxychlor D y = x R² = As / Ai As / Ai y = 5.829x R² = As / Ai As / Ai 1.5 Aldrin/Chlorpyrifos D10 60 y = x R² = Cs / Ci Cs / Ci 8 METHOD VALIDATION

22 METHOD LINEARITY, LOD, LOQ RT (min) Compou IS Chlorpyrifos D10 α-liane Chlorpyrifos D10 β-liane Chlorpyrifos D10 γ-liane Chlorpyrifos D10 δ-liane Chlorpyrifos D10 Heptachlor Chlorpyrifos D10 Aldrin Chlorpyrifos D10 Chlorpyrifos Heptachlor epoxide Chlorfenvinphos D10 Chlorfenvinphos D10 Chlorfenvinphos Chlorfenvinphos D10 Captan p,p-dde D8 γ-chlordane p,p-dde D8 α-eosulfan p,p-dde D8 α-chlordane p,p-dde D8 Bisphenol A p,p-dde D8 p,p-dde p,p-dde D8 Dieldrin p,p-ddt D8 Erin p,p-ddt D8 β-eosulfan p,p-ddt D8 p,p-ddd p,p-ddt D8 Erin Aldehyde p,p-ddt D8 p,p-ddt p,p-ddt D8 Eosulfan sulfate Methoxychlor D14 Erin ketone Methoxychlor D14 Methoxychlor Methoxychlor D14 Boscalid Linear R2 Conc Range (pg/ul) LOD (pg/ul) LOQ (pg/ul) Match Fi (*) Rev. Match Fi (*) Quant. Ion m/z:183 (SemiAutomatic) m/z:183 (SemiAutomatic) m/z:183 (SemiAutomatic) m/z:183 (SemiAutomatic) m/z:100 (SemiAutomatic) m/z:66 (SemiAutomatic) m/z:97 (SemiAutomatic) m/z:81 (SemiAutomatic) m/z:267 (SemiAutomatic) m/z:79 (SemiAutomatic) m/z:375 (SemiAutomatic) m/z:237 (SemiAutomatic) m/z:66 (SemiAutomatic) m/z:213 (SemiAutomatic) m/z:246 (SemiAutomatic) m/z:79 (SemiAutomatic) m/z:81 (SemiAutomatic) m/z:237 (SemiAutomatic) m/z:235 (SemiAutomatic) m/z:67 (SemiAutomatic) m/z:235 (SemiAutomatic) m/z:237 (SemiAutomatic) m/z:67 (SemiAutomatic) m/z:227 (SemiAutomatic) m/z:140 (SemiAutomatic) (*) evaluated at 131 ppb level METHOD VALIDATION

23 COMPARATIVE DATA GC-qMS GC GC-TOF Compou / IS Linearity Range (pg/ul) R2 LOQs (ng/g) LOQ (ng/g) α-bhc / α-eosulfan D4 β-bhc / α-eosulfan D4 γ-bhc / α-eosulfan D4 δ-bhc / α-eosulfan D4 Heptachlor / α-eosulfan D4 Aldrin / α-eosulfan D4 Chlorpyrifos /Chlorpyrifos D10 Heptachlor Epoxide /p,p'-dde D8 Chlorfenvinphos /Chlorfenvinfos D10 Captan /Captan D6 γ-chlordane /p,p'-dde D8 α-eosulfan / α-eosulfan D4 α-chlordane /p,p'-dde D8 Bisphenol A /Bisphenol A D16 p,p'-dde /p,p'-dde D8 Dieldrin /p,p'-dde D8 Erin /β-eosulfan D4 β-eosulfan /β-eosulfan D4 p,p'-ddd /p,p'-ddt D8 Erin aldehyde /p,p'-ddt D8 p,p'-ddt /p,p'-ddt D8 Eosulfan Sulfate /p,p'-ddt D8 Erin ketone /Methoxychlor D14 Methoxychlor /Methoxychlor D14 Boscalid /Methoxychlor D METHOD VALIDATION

24 REAL SAMPLES Sample F2/09 Stillbirth 40 weeks Male Liver tissue (500mg) TIC PRELIMINARY RESULTS

25 REAL SAMPLES Sample F2/09 Stillbirth 40 weeks Male Liver tissue (500mg) TIC RIC α-eosulfan 24ppb p,p-dde 5ppb PRELIMINARY RESULTS

26 REAL SAMPLES Sample F3/12 Stillbirth 37 weeks + 3 days Female Liver Tissue (500 mg) TIC PRELIMINARY RESULTS

27 REAL SAMPLES Sample F3/12 Stillbirth 37 weeks + 3 days Female Liver Tissue (500 mg) TIC RIC β-eosulfan 35ppb α-eosulfan 25ppb PRELIMINARY RESULTS

28 REAL SAMPLES Compou Quant. Ion Sample F2/09 (500mg) GC GC-TOF GC-qMS ng/g α-liane β-liane γ-liane δ-liane Heptachlor Aldrin Chlorpyrifos Heptachlor epoxide Chlorfenvinphos Captan γ-chlordane α-eosulfan α-chlordane Bisphenol A p,p-dde Dieldrin Erin β-eosulfan p,p-ddd Erin Aldehyde p,p-ddt Eosulfan sulfate Erin ketone Methoxychlor Boscalid m/z:183 (SemiAutomatic) m/z:183 (SemiAutomatic) m/z:183 (SemiAutomatic) m/z:183 (SemiAutomatic) m/z:100 (SemiAutomatic) m/z:66 (SemiAutomatic) m/z:97 (SemiAutomatic) m/z:81 (SemiAutomatic) m/z:267 (SemiAutomatic) m/z:79 (SemiAutomatic) m/z:375 (SemiAutomatic) m/z:237 (SemiAutomatic) m/z:66 (SemiAutomatic) m/z:213 (SemiAutomatic) m/z:246 (SemiAutomatic) m/z:79 (SemiAutomatic) m/z:81 (SemiAutomatic) m/z:237 (SemiAutomatic) m/z:235 (SemiAutomatic) m/z:67 (SemiAutomatic) m/z:235 (SemiAutomatic) m/z:237 (SemiAutomatic) m/z:67 (SemiAutomatic) m/z:227 (SemiAutomatic) m/z:140 (SemiAutomatic) Sample F3/12 (500mg) GC GC-TOF GC-qMS ng/g PRELIMINARY RESULTS

29 BENEFITS OF GC GC-TOF APPROACH Enhanced chromatographic separation Cryogenic peak compression Minimized matrix effect Minimized coelution of targets More reliable identification Improved LOD GC GC-TOF

30 CONCLUSION TOF-MS is delivering valuable analytical information for more reliable target identification at lower concentration compared to single quadrupole working in SIM mode To minimize the matrix effect on sensitivity, the GC GC approach is providing the highest degree of chromatographic separation capability, improving the LOD a maintaining good mass spectra quality at lower concentration level Preliminary results for unknown samples of human tissues, confirmed a better detectability by using GC GC-TOFMS, with the identification a quantitation of contaminants not visible with GC-qMS approach Mass spectra information will be of primary importance for possible further post-investigation of samples for different target a untargeted contaminants CONCLUSION

31 ACKNOWLEDGMENT Liquid Chromatography-Mass Spectrometry Laboratory of University of Urbino for the sample preparation method set up a samples extracts delivery Insitute of Patology Lino Rossi of the University of Milan for SIUD a SIDS victims autopsies samples Thank You!