Raising Confidence in the Analysis of Environmental Samples with Tandem Mass Spectrometry

Transcription

1 Raising Confidence in the Analysis of Environmental Samples with Tandem Mass Spectrometry Bruker Daltonics Chemical and Applied Markets Fremont, CA Ed George

2 Bruker s Portfolio of Chromatography and Mass Spectrometry Solutions GC GC/MS SQ GC/MS/MS TQ LC/MS/MS TQ LC/MS Ion Trap LC/MS TOF/QTOF MALDI TOF TOF/TOF FTMS ICP MS

3 Bruker s Portfolio of Chromatography and Mass Spectrometry Solutions Environmental Testing Targeted Quantitation & Confirmation Solutions GC GC/MS SQ GC/MS/MS TQ LC/MS/MS TQ Screening and Quant Solutions Microbiology and Conclusive Unknown ID Solutions Inorganic Contaminants LC/MS Ion Trap LC/MS TOF/QTOF MALDI TOF TOF/TOF FTMS ICP MS

4 Agenda The Process and Power of Multiple Reaction Monitoring (MRM) Key Applications for Tandem MS in Environmental Monitoring Pyrethroid Insecticides by EI and NCI with Tandem Mass Spectrometry PBDE Flame Retardants in Sewage and Ocean Sediments EPA Methods 8270 and 525: Raising Confidence in Results with MRM Analysis of PCPPs by LC/MS/MS

5 Environmental Monitoring: The Instrument Challenge Performance - Must meet performance requirements Sensitivity to meet regulatory limits Reality: Labs want to exceed those requirements Reliability Must be designed for Production Labs Data quality must be impeccable Consumer safety and profitability is at stake with every sample Robustness Withstand the Abuse of complex samples QuEChERs/8270 sludge extracts Hundreds of samples before required maintenance Ion source in LC-MS and GC-MS require reduced maintenance

6 Power of MRM in Environmental Matrices Product ion m/z MRM Transition S O P O O Parathion-Ethyl Precursor ion m/z O N O (mainlib) Parathion

7 Isobaric Interferences: Parathion Example q2 Q3 109 Q1 Parathion 291 Matrix Interference 291

8 Pumpkin matrix with 1ppb pesticide spike Scan range: 80Da to 400Da Full Scan 263 Da, search for halfenprox 263 Da??? Demeton-S-methyl Trifluralin Diazinon Heptachlor epoxide Endosulfan-sulfate MRM 263 > 235 Da Iprodione Paclobutrazol Halfenprox

9 Pyrethroid Insecticides by GC/MS/MS

10 Pyrethroid Facts Pyrethroids are used in household insecticides/repellents Are synthetic compounds based on structure of natural pyrethrins produced by the flowers of pyrethrums (Chrysanthemum cinerariaefolium and C. coccineum). Pyrethroids are toxic to fish and other aquatic organisms At extremely low levels, such as 2 parts per trillion, pyrethroids are lethal to mayflies, gadflies, and invertebrates that constitute the base of many aquatic and terrestrial food chains. Pyrethroids have been found to be unaffected by secondary treatment systems at municipal wastewater treatment facilities in California. They appear in the effluent, usually at levels lethal to invertebrates Therefore very low levels of detection are required in complex sediment and waste water effluent extracts

11 Pyrethroids- EPA and Other Methods 8270 is GC/MS full scan and/or SIM- no MSMS! 1660 is HPLC with UV Detection- Good luck! 1699 is HRGCMS method- Expensive! USGS method uses GC/MS and GC/MS/MS (EI)- Good and addresses some analytes that poorly dissociate in MRM GC/MS/MS in both EI and CI MRM modes were analyzed with the Bruker SCION for a specific group of pyrethroids Calibration and Instrument DLs determined followed by analysis of sewage and sediment sample extracts

12 Analytical Conditions - GC Analytical Conditions Parameter Setting Column BR-5ms, 15 m x 0.25 mm, 0.25 µm Gas Flow Helium, 1 ml/min Column Oven 60 ºC(hold 2min) 150 at 50 ºC/min(hold 1 min) 320 at 10ºC/min (hold 3 min) Injection 2 µl, pulsed splitless, 250ºC

13 Analytical Conditions MS- EI Analytical Conditions Parameter Setting Ion Source Temperature 250ºC Transferline Temperature 280ºC Collision Gas Measurement type Scan Times Argon (1.7 mtorr) Dynamic MRM Calculated by CBS

14 Analytical Conditions MS- NCI Analytical Conditions Parameter Setting Ion Source Temperature 150ºC Transferline Temperature 250ºC Collision Gas Reagent Gas CI Gas Pressure Measurement type Scan Times Argon (1.7 mtorr) Ammonia 15 psi Dynamic MRM Calculated by CBS

15 EI MRM Transitions Compound Name Q1 Mass Q3 Mass trans-permethrin trans-permethrin trans-permethrin Prallethrin Prallethrin Fipronil Sulfide Esfenvalerate-B Esfenvalerate-B Esfenvalerate-B Allethrin Allethrin Fipronil-sulfone Fipronil-sulfone Danitol Danitol Bifenthrin Bifenthrin Bifenthrin Cyfluthrin isomers Cyfluthrin isomers Cyfluthrin isomers Cypermethrin isomers Cypermethrin isomers Collision Energy Deltamethrin Deltamethrin Deltamethrin Esfenvalerate-A Esfenvalerate-A Esfenvalerate-A Fipronil Fipronil Fipronil cis-permethrin cis-permethrin cis-permethrin Piperonylbutoxide Piperonylbutoxide Piperonylbutoxide Cyhalothrin Cyhalothrin

16 NCI MRM Transitions Compound Name Q1 Mass Q3 Mass Collision Energy cis-permethrin cis-permethrin trans-permethrin trans-permethrin Fipronyl sulfide Fipronyl sulfide Fipronyl sulfide Esfenvalerate-B Esfenvalerate-B Fipronyl sulfone Fipronyl sulfone Bifenthrin Bifenthrin Cypermethrin isomers Cypermethrin isomers Deltamethrin Deltamethrin Deltamethrin Deltamethrin Allethrin Danitol Cyfluthrin isomers Cyfluthrin isomers Esfenvalerate-A Esfenvalerate-A Pallethrin

17 Good Isomer Separation on 15 M column EI CI

18 EI-MRM Calibration ppb Compound Name Corr. Avg. RRF % RSD Fipronil Sulfide Allethrin Prallethrin Fipronil-sulfone Fipronil Piperonylbutoxide Bifenthrin Danitol Cyhalothrin cis-permethrin trans-permethrin Cyfluthrin isomers Cypermethrin isomers Esfenvalerate-A Esfenvalerate-B Deltamethrin Mean

19 EI-MRM IDLs Parameter Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 mean sd Calculated IDL Target concentration % RSD Fipronil Sulfide Allethrin Prallethrin Fipronil-sulfone Fipronil Piperonylbutoxide Bifenthrin Danitol Cyhalothrin cis-permethrin trans-permethrin Cyfluthrin isomers Cypermethrin isomers Esfenvalerate-A Esfenvalerate-B Deltamethrin Mean

20 NCI-MRM Calibration ppb Compound Name Corr. Avg. RRF % RSD Fipronyl sulfide Allethrin Pallethrin Fipronyl sulfone Fipronil Bifenthrin Danitol Cyhalothrin cis-permethrin trans-permethrin Cyfluthrin isomers Cypermethrin isomers Esfenvalerate-A Esfenvalerate-B Deltamethrin Average

21 NCI-MRM Low Calibration ppt Compound Name Corr. Avg. Resp % RSD Fipronyl sulfide Fipronyl sulfone Bifenthrin Danitol Esfenvalerate-A Esfenvalerate-B Deltamethrin Average

22 NCI-MRM IDLs Parameter Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 mean sd Calculated IDL Target concentration % RSD Fipronil Sulfide Allethrin Prallethrin Fipronil-sulfone Bifenthrin Danitol Cyhalothrin cis-permethrin trans-permethrin Cyfluthrin isomers Cypermethrin isomers Esfenvalerate-A Esfenvalerate-B Deltamethrin Fipronil Mean

23 NCI-MRM Low-Level IDLs (Units in ppt) Parameter Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 mean sd Calculated IDL Target concentration Fipronil Sulfide Fipronil-sulfone Bifenthrin Danitol Esfenvalerate-A Esfenvalerate-B Deltamethrin Fipronil % RSD Mean

24 NCI Sensitivity Excellent for Select Cmpds 1 ppt Fipronyl Sulfide 10 ppt Bifenthrin

25 NCI-MRM: Excellent vs. Matrix Ocean Sediment Spike Sewage Effluent Spike Cal Standard

26 EI-MRM: Matrix effects results Ocean Sediment Sewage Effluent Cal Standard

27 EI-MRM: Matrix Closer Look Ocean Sediment Ocean Sediment Sewage Sewage Cal standard Cal standard

28 NCI: Allethrin/Pallethrin Poorly dissociates in MS/MS (m/z 167) however parent to parent with Ar in cell cleans up the matrix nicely Ocean Sediment Sewage Cal Standard

29 EI-MRM: Allethrin/Pallethrin Extreme matrix interference at MRM transitions studied! Ocean Sediment Sewage Cal Std

30 Deltamethrin- Both Techniques EI-MS/MS NCI-MS/MS Ocean Sediment Sewage Cal Standard

31 Pyrethroid Analysis- Lessons Learned MS/MS will provide good confidence in your data, at extreme low levels, however. Consider your test matrix and added discrimination using NCI, especially approaching sub ppb levels and very dirty extracts Poorly dissociated cmpds can still benefit parent-toparent transitions vs. SIM alone! Not all cmpds will work well by NCI, look at structure and optimize conditions Watch out for matrix enhanced chromatographic response high bias NOT due to detection technique but rather matrix shielding of active sites in injection and chromatographic pathway

32 Brominated Flame Retardants by GC/MS/MS

33 PBDE Facts Used as flame retardants in a variety of products Tend to bio-accumulate in tissues, breast milk Shown to be related to increased infertility rates in humans at levels that are normally found in US households Studies in Canada have found significant concentrations of PBDEs in common foods such as salmon, ground beef, butter, and cheese PBDEs have also been found at high levels in indoor dust, sewage sludge, and effluents from wastewater treatment plants. Increasing PBDE levels have been detected in the blood of marine mammals such as harbor seals.

34 PBDEs- Method Considerations EPA 1614A is HRGCMS- Expensive! EPA 8081 is GC with ECD Detection- Background! EPA 527 is GCMS method- No MSMS! USGS method uses GC/MS and GC/MS/MS (EI) GC/MS/MS in EI MRM mode was analyzed with the Bruker SCION for a specific group of PBDEs Calibration and Instrument DLs determined followed by analysis of sewage and sediment sample extracts

35 Analytical Conditions - GC Analytical Conditions Parameter Setting Column BR-5ms, 15 m x 0.25 mm, 0.25 µm Gas Flow Helium, 1 ml/min Column Oven 60 ºC(hold 2min) 150 at 50 ºC/min(hold 1 min) 325 at 10ºC/min (hold 8 min) Injection 2 µl, pulsed splitless, 290ºC

36 Analytical Conditions MS- EI Analytical Conditions Parameter Setting Ion Source Temperature 250ºC Transferline Temperature 280ºC Collision Gas Measurement type Scan Times Argon (1.7 mtorr) Dynamic MRM Calculated by CBS

37 MRM Transitions Compound Name Q1 Mass Q3 Mass BDE BDE SS-BDE-209-C SS-BDE-209-C BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE SS-BDE-4FHBE BDE BDE Collision Energy BDE BDE IS-BDE-4FTBE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE

38 TIC MRM Chromatogram- 50 ppb std

39 MRM Calibration ppb Compound Name Corr. Avg. RRF % RSD BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE SS-BDE-4FHBE BDE BDE BDE SS-BDE-209-C BDE Mean

40 MRM IDLs Parameter Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 mean sd Calculated IDL Target concentration % RSD BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE SS-BDE-4FHBE BDE BDE BDE SS-BDE-209-C BDE Mean

41 TIC MRM in Matrix-1 Ocean Sediment Sewage extract CAL Standard

42 TIC MRM in Matrix-2 Ocean Sediment Sewage Extract CAL Standard

43 TIC MRM in Matrix-3 Ocean Sediment BDE-209 Sewage Extract CAL standard

44 PBDE Analysis by GC/MS/MS MS/MS provides excellent discrimination against the matrices studied to provide good confidence in your data Be aware however of matrix enhanced chromatographic response BDE-209 sensitivity can be improved with thinner phase and even shorter column to reduce elution temperature by minimizing thermal degradation

45 EPA Methods 8270 and 525: Raising Confidence in Results with Tandem Mass Spectrometry

46 Method 8270: Want Lower Detection Levels? Method is for a wide variety of compounds in complex matrices Contractors and labs may want lower reporting limits than method was validated for Labs can use SIM, but what if isobaric interferences in heavy matrix? MS/MS can give added specificity and confidence in results at low levels of detection

47 8270 Test Sludge Extract PCP injection 1 PCP injection after 35 Left- Blank Extract; Right-Sludge Extract

48 Sludge Sample Injection: Full Scan TIC of highly contaminated soil extract from EPA The sample was spiked with 100 pg per component for precision testing by Tandem Mass Spectrometry

49 MS/MS to Find Needles in the Haystack MS/MS on same Extract Previous Slide MS/MS on SCION in soil extract, pg injected on column pulsed-split injection

50 Raw Peak Area Raw Peak Area Raw Peak Area Method Precision Examples: 100 Injections at 100 ppb in Sludge Extract 1-Chloronaphthalene %RSD = 5.7% 2,4-Dichlorophenol %RSD = 4.7% Series Series Injection Number Injection Number 4-Bromophenoxy Ether %RSD = 6.6% Injection Number Series1

51 Method 525: Drinking Water (DW) Method Method covers wide variety of pesticides and industrial pollutants Current version written as Full Scan with SIM target analytes for lower reporting limits Method applies to finished DW; however, often used to test source waters as in past method version (525.2) (ie. Surface and Ground Waters) Method concentrates analytes (and matrix) by SPE and inject 1-2 ul into GC/MS MS/MS can give better specificity and confidence in results at low levels of detection

52 Tandem MS and SIM in STD Look the Same A B TIC of over one hundred compounds in EPA in MS/MS (A) and SIM (B) acquisition modes at concentration of 500 ng/ml in a pure solvent.

53 But in Surface Water Extracts FULL SCAN Actual surface water source sample submitted for Method 525 analysis. Many water treatment plants are required to monitor source waters for regulated and unregulated contaminants.

54 Chromatograms Showing d-bhc in Surface Water Extract SIM mode in solvent MRM mode in solvent SIM mode in matrix MRM mode In matrix

55 Chromatograms Showing Triademefon in Surface Water Extract SIM mode in solvent MRM mode in solvent SIM mode in matrix MRM mode In matrix

56 SIM vs. MS/MS Recovery Comparisons Spiked in SW measured by SIM Spiked SW measured by MRM Peak Name RT (min) Measured %RSD Measured %RSD Chlorprophan a-bhc b-bhc g-bhc d-bhc ND ND Metribuzin MGK Endosulfan II Average Note: Spiked in the matrix at 50 ng/ml (n=5)

57 Conclusions Full Scan and SIM are techniques required in Methods 8270 and 525 However, lower reporting limits and heavy matrix challenge this requirement Tandem MS provides lower detection limits and confidence in results, eliminating isobaric interferences and potential erroneous reporting (highbiased)

58 Analysis of Trace PPCPs in Water Using The EVOQ TQ LC-MS/MS System with OLE

59 Bruker EVOQ With OLE for PPCPs in Water PPCPs refers to a broad group of chemical substances used for health or cosmetic reasons, or used by agribusiness to boost growth or health of livestock PPCPs= Pharmaceuticals and Personal Care Product Wastewater from households and industrial sources contains multiple PPCPs of various combinations, concentrations and relative toxicologies PPCPs are bioactive, they do not dissolve easily or evaporate at normal temperatures or pressures, so can easily enter the soil and aquatic environments via sewage, biosolids and irrigation Evidence suggests PPCPs are linked to some ecological damage such as the delayed development in fish and delayed metamorphosis in frogs Conventional methods of PPCP detection requires the pre-concentration of large volume water samples and tedious solid phase extraction (SPE) clean up to achieve the low ng/l (ppt) level detection This study demonstrates how using UHPLC with an integrated on-line extraction (OLE) option coupled to LC-MS/MS can be used for the detection of PPCPs The on-line extraction module enables convenient method-driven on-line sample cleanup or sample pre-concentration

60 Bruker EVOQ With OLE for PPCPs in Water Experimental Conditions Chromatography parameters (Advance UHPLC OLE) Trap column YMC-Pack ODS-AQ, 3µm, 35 mm x 2.0 mm I.D. Column temperature 40 C Injection volume 400 µl Flow rate 400 µl/min Solvent A 2 mm ammonium formate, 0.1% FA in water Solvent B 2 mm ammonium formate, 0.1% FA in MeOH Mass spectrometer parameters (EVOQ Elite) HV 4000 V Cone gas flow 15 units Cone gas temperature 300 C Heated probe gas flow 40 units Heated probe temperature 450 C Nebulizer gas flow 50 units Exhaust gas On Q2 pressure 1.5 mtorr (Argon) Solvent C Gradient conditions 2 mm ammonium formate, 0.1% FA in water 0.0 min, 10% B 0.2 min, 10% B 0.8 min, 25% B 8.0 min, 95% B 9.0 min, 95% B 9.1 min, 10% B 12.0 min, 10% B Sample Preparation: Water sample acidified with 0.1% FA and filtered (0.45um)

61 Bruker EVOQ With OLE for PPCPs in Water Target Compounds

62 Bruker EVOQ With OLE for PPCPs in Water Selected PPCPs at 2 ppt

63 Bruker EVOQ With OLE for PPCPs in Water Linearity, 1 ppt 100 ppt Trimethoprim R 2 =0.999 Carbamazepine R 2 =0.999 Naproxen R 2 =0.999 Triclosan R 2 =0.998 Gemfibrozil R 2 =0.997 Sulfamethoxazole R 2 =0.999

64 Bruker EVOQ With OLE for PPCPs in Water Summary of PPCPs Calibration Curves Compound Name RT S/N (min) ESI R 2 (p/p) 2 ppt RSD n=10 (5ppt) Trimethoprim % Hydroxy Atrazine % Thiabendazole % Ciproxacin % Caffeine % Sildenafil % Sulfamethoxazole % Cyanazine % Simazine % Metribuzin % Hexazinone % Dapoxetine % Bentazone % Ametryn % Carboxine % Carbamazepine % Atrazine % Alpazolam % Diuron % Prometryn % 2,4-D % MCPA % Mecoprop % Metolachlor % Pyriproxifen %

65 Bruker EVOQ With OLE for PPCPs in Water Summary of PPCPs in Water Samples Compound Name Creek Bottle Tap Water 1 Tap Water 2 Water Water pg/ml (ppt) Trimethoprim <2 <2 5 <2 Hydroxy Atrazine 4 <2 7 <2 Thiabendazole ND <2 <2 <2 Ciproxacin ND ND ND ND Caffeine ND <2 <2 10 Sildenafil ND ND ND <2 Sulfamethoxazole <2 <2 ND <2 Cyanazine ND ND ND <2 Simazine 3 <2 5 ND Metribuzin ND ND ND ND Hexazinone ND Dapoxetine ND ND ND ND Bentazone ND ND ND ND Ametryn ND ND <2 ND Carboxine ND ND ND ND Carbamazepine <2 <2 <2 ND Atrazine <2 ND ND ND Alpazolam ND ND ND ND Diuron 9 <2 6.2 ND Prometryn ND ND ND <2 2,4-D 9 <2 13 <2 MCPA <2 <2 <2 ND Mecoprop <2 < Metolachlor 22 <2 <2 <2 Pyriproxifen ND <2 ND <2

66 Bruker EVOQ With OLE for PPCPs in Water Summary All of the PPCPs studied were detected at 2 ppt or better with an injection of 400 ul water samples, with a linear response range up to 200 or 500 ppt. Replicate injections with 5 ppt level spiked in tap water demonstrated excellent robustness This new method illustrates how modern UHPLC with OLE and LC-MS/MS can provide the sensitivity demanded by regulatory bodies, even with challenging ranges of samples at various concentrations The technique also presents a more convenient and simpler approach to the analysis of PPCPs compared to traditional solid phase extraction techniques

67 Conclusions The power of Multiple Reaction Monitoring (MRM) in Environmental Samples Can reduce detection/reporting limits and provide more confidence in your data Enables matrix discrimination in samples against isobaric interferences that will occur with SIM alone Other techniques such as NCI as shown in pyrethroids may be required to eliminate matrix interferences Keep in mind that your matrix needs some study for optimized MRM transitions and/or ionization techniques for best results