Accurate Mass Spectrometer to Expand the Scope of EPA ......Octadecyl (C-18) functional group after adding Surrogates. The eluent from SPE is concentrated to dryness, and then diluted
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Use of a High Resolution Accurate Mass Spectrometer to Expand the Scope of EPA
Methods for Emerging Contaminants (EPA 539)
October 2015
Andy Eaton, PhD, BCESTechnical Director
Ali Wolfgang HaghaniTechnical R&D Manager
Eurofins Eaton Analytical Monrovia, CA 91016
www.eatonanalytical.com
Hormones as Emerging Contaminants (EC) is NOT a New Concept
•~1950 as the TOF-MS and GC-MS concepts were introduced at Harvard, scientists were already speculating on fate and transport of hormones in water streams.
• ~ 1970-80’s New analytical technologies made studies possible using GC-MS or TOF-MS but still detection limits were inadequate for needed levels.
• 2001 USGS; Koplin et al. published their Water-Quality Data for Pharmaceuticals, Hormones, and Other Organic Wastewater Contaminants in U.S. Streams, 1999-2000 - some at low ppt levels, using LCMS as SIM for two of their 5 methods.
• 2010 EPA 539 by Glynda Smith et al. was published for UCMR 3 monitoring (2013-2015) using Tandem MS for selected hormones at low to sub ppt levels. The lowest reporting limit was set for Testosterone at 0.1ppt.
•Directive 2013/39/EU (Europa made of 28 nations) – Estrone, Estradiol and Ethynylestradiol at 1.0, 0.3 and 0.03ppt in water streams such as effluents.
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2011 Q Exactive: a Hybrid HRAM is Added to the Orbi Exactive Family
3From Trussell, Clarke Prize Lecture 2013
2005 LTQ2007 Exactive2011 Q Exactive…2015 PLUS & HF
EPA 539 summary
MS requirements of the method:Summary : A 1 Liter of dechlorinated sample with Omadine as a biocide is extracted through SPE Octadecyl (C-18) functional group after adding Surrogates. The eluent from SPE is concentrated to dryness, and then diluted to 1 ml with 50/50% methanol/water. An aliquot is injected to the LC-MS/MS after adding internal standards and quantified against the IS.
3.16 MULTIPLE REACTION MONITORING (MRM) – A mass spectrometric technique in which a precursor ion is first isolated, then subsequently fragmented into a product ion(s). Quantitation is accomplished by monitoring a specific product ion. As described in Section 10.1.2, MS parameters must be optimized for each precursor ion (Sect. 3.17) and product ion (Sect. 3.19).
6.12.3 ELECTROSPRAY IONIZATION-TANDEM MASS SPECTROMETER (ESI-MS/MS) – The mass spectrometer must be capable of rapid switching between negative ion and positive ion electrospray ionization modes. The system must be capable of performing MS/MS to produce unique product ions (Sect. 3.19) for the method analytes within specified retention time segments. At least 10 – 15 scans across the chromatographic peak are needed to ensure adequate precision.
Orbitrap™Technology meets these requirements
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Why Consider Orbitrap Technology for Method 539? We are becoming more and more concerned
about “non-target” analytes. Traditional LC-MS/MS systems are focused
entirely on target analytes. Orbitrap and Q-TOF (time of flight) sensitivity has
improved over the last decade Orbitrap has resolution of >70,000, allowing one to
also watch for false positives and false negatives. The UCMR 3 database shows a lot (>5% of PWS) of
“sporadic” results for hormones. It is important to ensure they are real.
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Evaluating the QE in Parallel Reaction Mode (PRM) for EPA 539 Initial Demonstration of capability (IDC) from the method: 1. DEMONSTRATION OF LOW SYSTEM BACKGROUND <1/3 MRL 2. DEMONSTRATION OF PRECISION – 4-7 LFB’s RSD </= 20% 3. DEMONSTRATION OF ACCURACY – LFB’s recovery diff. </= 30% of T.V. 4. MINIMUM REPORTING LEVEL (MRL) CONFIRMATION or doing LCMRL
Targeted compounds and their MRLs:Analyte DL Fortified Concentration (ng/L) DL (ng/L) LCMRL (ng/L)Estriol 1.10 0.24 0.28Estrone 1.05 0.19 4.017β-Estradiol 1.30 0.39 0.3217α-Ethynylestradiol 1.75 0.33 1.3Androstenedione 0.50 0.20 0.37Testosterone 0.50 0.04 0.062Equilin 1.25 2.94 3.0
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EPA 539 Is Written For LC-MS/MS So The QE PRM Mode Is Truest To The Method
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Quadrupole-equipped HR/AM instrumentsHR/AM analyzer permits parallel detection of all target product ions in one concerted high resolution mass analysis
Serial monitoring
Parallel monitoring
Drag And Drop Method Development For NEG/POS/NEG On The Same Run for EPA 539
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All Calibration Coefficients > 0.99 and Good Dynamic Range and Linearity
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EE2 range 0.225 – 7.2ppt
An Example of a Calibration for EE2 Using TraceFinder 3.2
Compound Calibration Report
Lab Name: EEA Method:021215-539-PRM-IDOC-LINEAR_EPA-539-PRMInstrument: Q EXACTIVE EPA-539-PRMUser: Thermo Cali File:021215-539-PRM-IDOC-LINEAR.calx
Batch:021215-539-PRM-IDOC-LINEAR
Compound Name: 17 alpha-Ethynylestradiol
LinearPass
LevelStd
Amount Std Area IS Amount IS AreaResp factor/
ratio
CalcAmount Units % CV % RSD
0 0 419 100 9244925 0 0.002 ng/L N/A N/A
ICAL-1 0.225 63224 100 10063239 0.006 0.231 ng/L N/A N/A
ICAL-2 0.45 110807 100 9324158 0.012 0.436 ng/L N/A N/A
ICAL-3 0.9 243598 100 9755408 0.025 0.915 ng/L N/A N/A
ICAL-4 1.8 491433 100 9920757 0.05 1.815 ng/L N/A N/A
ICAL-5 3.6 942018 100 9557799 0.099 3.612 ng/L N/A N/A
ICAL-6 5.4 1415265 100 9788761 0.145 5.298 ng/L N/A N/A
ICAL-7 7.2 1850842 100 9334500 0.198 7.266 ng/L N/A N/A
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Determination of Minimum Reporting Limit (MRL) using LCMRL calculation
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The LCMRL is defined as the lowest spiking concentration at which recovery of between 50 and 150 percent is expected 99 percent of the time by a single analyst. The procedure requires, at a minimum, four replicates at each of seven fortification levels. Four laboratory reagent blanks should also be included. All must be processed through the entire method procedure
http://water.epa.gov/scitech/drinkingwater/labcert/analyticalmethods_ogwdw.cfm
NB – Calculator only works with 32 bit computers.
LCMRL<0.19 ppt For EE2. It Could Not Be Calculated Because It Needed A Lower Point
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0.05ppt as critical level
Area and Height of the Lowest CAL Stdfor EE2 – Using Trace Finder 3.2
Compound Name: 17 alpha-EthynylestradiolSample Conc: 0.225 ng/LRetention time: 14.96Area (Quan): 61535Height (Quan): 5855
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Can Directive 2013/39/EU be achieved at 0.03ppt based on Area & Height ?
HRAM is MUCH More Sensitive than the Published LCMRL for EPA539Laboratory: Eurofins Eaton Analytical - EEA
Extraction: Concentrated 500 ml of solutions to final 1 ml - per EPA 539 preservatives/extraction procedure
Instrument: Q- Exactive, hybrid quadrupole Mass Spectrometry HRAM,Resolution for collected data : 70000 (mz 200 FWHM)AGC 2e5Max IT 200 msIsolation window 1.0 m/z
Scans/peak collected >10-15 scans per peaks
HPLC: Thermo RS Ultimate UHPLC 3000, binary pump, autosampler and column heater with 100 ul sample loopColumn: Acclaim Polar Advantage II, 2.1x150 mm, 3 um, 120AEluents: A) 1 mM Ammonium Fluoride B) 50%/50% ACN/MeoH
Gradient flow at 0.3ml/min with a 21.4 minutes run.Injection volume: 50 ul
Method 539 UCMR3 Proposed Analyte UCMR MRL (ng/L) EPA 539 published LCMRL
(ng/L)
Q Exactive HRAM 70000 Res.
LCMRL (ng/L)
QE-LCMRL Calc -DL (ng/L)
17α-ethynylestradiol 0.9 1.3 Critical level 0.05 0.117β-estradiol 0.4 0.32 0.17 0.047equilin 4 0.28 Critical level 0.23 0.48estriol 0.8 3 0.27 0.2estrone 2 4 0.84 0.48testosterone 0.1 0.062 0.033 0.0274-androstene-3,17-dione 0.3 0.37 0.19 0.08
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This got EPA (Glynda Smith’s) one thumb up of approval and, as she predicted, it was much more sensitive.
Precision and Accuracy for LFB (10xMRL) and a Spiked Sample at MRL
QE-HRAM-MS2 70000 resolution PRECISION AND ACCURACY IN FORTIFIED REAGENT WATER (n=4)Analyte Fortified Concentration (ng/L) Avg. %Recovery %RSD17α-ethynylestradiol 7.2 82% 417β-estradiol 3.2 84% 3equilin 32 81% 3estriol 6.4 100% 4estrone 16 83% 4testosterone 0.8 87% 5
4-androstene-3,17-dione 2.4 85% 8
QE-HRAM-MS2 70000 resolution PRECISION AND ACCURACY IN Sample 20150108002 (2) Low Level spikeAnalyte Fortified Concentration (ng/L) Avg. %Recovery %RSD17α-ethynylestradiol 0.72 95% 2.317β-estradiol 0.32 87% 0.6equilin 3.2 92% 8.2estriol 0.64 101% 4.0estrone 1.6 95% 3.4testosterone 0.08 99% 0.1
4-androstene-3,17-dione 0.24 118% 0.1
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Continued: Spiked Field Samples at Reporting Levels. Samples Had No Hits.
QE-HRAM-MS2 70000 resolution PRECISION AND ACCURACY IN Sample 20150108002 (2) Low Level spikeAnalyte Fortified Concentration (ng/L) Avg. %Recovery %RSD17α-ethynylestradiol 0.72 95% 2.317β-estradiol 0.32 87% 0.6equilin 3.2 92% 8.2estriol 0.64 101% 4.0estrone 1.6 95% 3.4testosterone 0.08 99% 0.14-androstene-3,17-dione 0.24 118% 0.1
QE-HRAM-MS2 70000 resolution PRECISION AND ACCURACY IN Sample 201501090001 (2) Low Level spikeAnalyte Fortified Concentration (ng/L) Avg. %Recovery %RSD17α-ethynylestradiol 0.72 97% 1617β-estradiol 0.32 95% 13equilin 3.2 92% 11estriol 0.64 107% 13estrone 1.6 95% 14testosterone 0.08 97% 194-androstene-3,17-dione 0.24 104% 20
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This got Glynda Smith’s second thumbs up. Matrix spike recovery at the MRL is a truer measure of performance than the LCMRL.
Second Approach - How About Looking For Non Targeted And Unknowns While Quantifying For EPA 539 Compounds Simultaneously ?
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DIA # 1: 50 amuisolation window, loop 6DIA # 2: Estriolwith isolation window 1 amu, NCE 75Loop 1DIA # 3: 50 amuisolation window loop 1DIA # 4 : 200 amuisolation window loop 2
Why adding a PRM for estriol?
Estriol Problematic For the Full-MS (70K) Method.No Signal Collected for Cal1&2 (0.2 & 0.4ppt) Stds
CAL 1 & 2ICAL-3 = MRL
Estriol At Ultra Low Concentrations Shows Selectivity Issues @ 70000 res(TSIM/ddMS2)
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5 ppm accuracy filter
Internal Std Estriol-d2 Spiked At 10x In DI 289.7773 Amu And Contaminated Interference 287.16171 As A Suspect Causing Non Selectivity >10ppm For Estriol 287.16528
C:\TraceFinderData\...\Data\030515-071 03/25/15 20:59:58 IS
RT: 5.78 - 8.09 SM: 5B
6.0 6.5 7.0 7.5 8.0Time (min)
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6.61
6.197.657.22 7.30
NL: 1.73E7TIC F: FTMS - p ESI Full ms2 287.88@hcd56.67 [50.00-325.00] MS 030515-071
RT: 5.78 - 8.09 SM: 5B
6.0 6.5 7.0 7.5 8.0Time (min)
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6.61
6.197.657.22 7.30
NL: 1.73E7TIC F: FTMS - p ESI Full ms2 287.88@hcd56.67 [50.00-325.00] MS 030515-071
030515-071 #3243 RT: 6.61 AV: 1 NL: 1.82E7F: FTMS - p ESI Full ms2 287.88@hcd56.67 [50.00-325.00]
285 286 287 288 289 290 291 292 293 294m/z
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289.17773R=16706
290.18115R=16702
288.17154R=15502
291.18369R=15002
287.16171R=15102
030515-071 #3243 RT: 6.61 AV: 1 NL: 2.84E5F: FTMS - p ESI Full ms2 287.88@hcd56.67 [50.00-325.00]
286.6 286.8 287.0 287.2 287.4 287.6 287.8 288.0m/z
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287.16171R=15102
Possible interfering unknown
Full MS Does Not See 287.16528. DIA/PRM Sees Precursors In MS2 Plus The Product Ion – Spiked Sample At MRL
C:\TraceFinderData\32\DIA-4 05/16/15 18:16:48
RT: 4.90 - 7.67 SM: 5B
5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6Time (min)
50
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Re
lative
Ab
un
da
nce
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1005.935.91 5.965.79 6.05 6.13 6.386.33 6.445.77 6.475.575.40 6.555.465.325.26 6.876.645.74 7.045.23 6.93 7.10 7.13 7.21 7.335.62 7.415.15 7.565.125.03
7.02 7.086.99 7.116.946.596.56 6.62 6.766.37 7.166.34 7.28 7.34 7.42 7.456.25 7.576.175.975.945.775.755.69
7.067.03 7.09
7.116.977.146.94 7.176.91 7.266.636.57 6.80 7.607.557.406.406.345.225.16
5.50 5.945.33 6.005.895.445.27 5.80 6.035.24 5.55 5.58 6.14 6.205.16 6.31 6.42 6.484.99 6.565.63 6.65 7.056.976.85 7.11 7.14 7.22 7.31 7.54 7.57
NL: 1.21E6Base Peak m/z= 287.16383-287.16671 F: FTMS - p ESI Full ms [100.00-1000.00] MS DIA-4
NL: 9.44E4Base Peak m/z= 287.16383-287.16671 F: FTMS - p ESI Full ms2 275.00@hcd55.00 [50.00-315.00] MS DIA-4NL: 7.70E3Base Peak m/z= 287.16383-287.16671 F: FTMS - p ESI Full ms2 287.17@hcd75.00 [50.00-310.00] MS DIA-4NL: 1.23E5Base Peak m/z= 287.16383-287.16671 F: FTMS - p ESI Full ms2 325.00@hcd55.00 [50.00-365.00] MS DIA-4
DIA-4 #2881 RT: 7.01 AV: 1 NL: 6.81E5T: FTMS - p ESI Full ms [100.00-1000.00]
286.98 287.00 287.02 287.04 287.06 287.08 287.10 287.12 287.14 287.16 287.18 287.20 287.22 287.24 287.26 287.28m/z
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287.16611
287.12924
287.22327
287.14124 287.18661 287.20203287.09473
C:\TraceFinderData\32\DIA-4 05/16/15 18:16:48
RT: 4.90 - 7.67 SM: 5B
5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6Time (min)
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1005.935.91 5.965.79 6.05 6.13 6.386.33 6.445.77 6.475.575.40 6.555.465.325.26 6.876.645.74 7.045.23 6.93 7.10 7.13 7.21 7.335.62 7.415.15 7.565.125.03
7.02 7.086.99 7.116.946.596.56 6.62 6.766.37 7.166.34 7.28 7.34 7.42 7.456.25 7.576.175.975.945.775.755.69
7.067.03 7.09
7.116.977.146.94 7.176.91 7.266.636.57 6.80 7.607.557.406.406.345.225.16
5.50 5.945.33 6.005.895.445.27 5.80 6.035.24 5.55 5.58 6.14 6.205.16 6.31 6.42 6.484.99 6.565.63 6.65 7.056.976.85 7.11 7.14 7.22 7.31 7.54 7.57
NL: 1.21E6Base Peak m/z= 287.16383-287.16671 F: FTMS - p ESI Full ms [100.00-1000.00] MS DIA-4
NL: 9.44E4Base Peak m/z= 287.16383-287.16671 F: FTMS - p ESI Full ms2 275.00@hcd55.00 [50.00-315.00] MS DIA-4NL: 7.70E3Base Peak m/z= 287.16383-287.16671 F: FTMS - p ESI Full ms2 287.17@hcd75.00 [50.00-310.00] MS DIA-4NL: 1.23E5Base Peak m/z= 287.16383-287.16671 F: FTMS - p ESI Full ms2 325.00@hcd55.00 [50.00-365.00] MS DIA-4
DIA-4 #2888 RT: 7.03 AV: 1 NL: 1.50E4F: FTMS - p ESI Full ms2 287.17@hcd75.00 [50.00-310.00]
170.60 170.65 170.70 170.75 170.80 170.85 170.90 170.95 171.00 171.05 171.10 171.15 171.20 171.25 171.30 171.35m/z
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171.08087
171.04462171.11725
Product 171.0808
Precursor-287.16528
2.8ppm
PRM PRM
DIA
DIA DIA
DIA
Full Full
Estriol Lowest Standard Using Trace Finder 3.2 Spectral Library Match / Identified
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Full-MS/DIA gives the same result as PRM experiment. No selectivity issues in our experimental set up or we have not gone low enough to test it. Precision/Accuracy of LFB and Matrix spikes matched very closely to PRM experiment.
Extraction: Concentrated 500 ml of solutions to final 1 ml - per EPA 539 preservatives/extraction procedure
Instrument: Q- Exactive, hybrid quadrupole Mass Spectrometry HRAM, Q- Exactive, hybrid quadrupole Mass Spectrometry HRAM,
Resolution for collected data : 70000 (mz 200 FWHM)Full-MS - Resolution for collected data : 70000 (mz@200 FWHM)
AGC 2e5DIA 17500 (mz@200FWHM)
Max IT 200 ms Estriol PRM as a line with isolation window 1 amu NCE=75
Isolation window 1.0 m/zDIA experiment one 100-520 60 amu isolation window, loop 7DIA experiment #2 510-1100 200 amu isolation window, loop 2
HPLC: Thermo RS Ultimate UHPLC 3000,, binary pump, autosampler and column heater with 100 ul sample loop Step NCE 35, 80
Column: Acclaim Polar Advantage II, 2.1x150 mm, 3 um, 120A
Eluents: A) 1 mM Ammonium Fluoride B) 50%/50% ACN/MeoH
Gradient flow at 0.3ml/min with a 21.4 minutes run.
Injection volume: 50 ul
PRM PRMFULL-
MS/DIA(EstriolPRM)
FULL-MS/DIA(Estriol
PRM)
Method 539 UCMR3 Proposed Analyte UCMR MRL (ng/L) EPA 539 published LCMRL
(ng/L)
Q ExactiveHRAM 70000 Res. LCMRL
(ng/L)
QE-LCMRL Calc -DL (ng/L)
Q Exactive HRAM 70000 Res. LCMRL
(ng/L)
QE-LCMRL Calc -DL (ng/L)
17α-ethynylestradiol 0.9 1.3Critical level
0.05 0.1 Critical level 0.05 0.1
17β-estradiol 0.4 0.32 0.17 0.047 0.16 0.12
equilin 4 0.28Critical level
0.23 0.48 Critical level 0.23 0.44
estriol 0.8 3 0.27 0.2 Critical level 0.046 0.068estrone 2 4 0.84 0.48 0.7 0.45testosterone 0.1 0.062 0.033 0.027 NA NA
4-androstene-3,17-dione 0.3 0.37 0.19 0.08 NA NA23
Using Full-MS data with Sieve Software for Site Specific Emerging Contaminants
FrameTable: 209 Items CompID ID CompMW Formula MZ Time HitCount
10 10 211.1428815 C9H24N1[32]S2 210.1356049 5.512476921 1
DetailTable: 1 Items
CSID Name Formula SMILES MW AvgMass MonoisotopicMass SearchMass DeltaPPM Adduct
14620 Atraton C_{9}H_{17}N_{5}O CC/N=c/1\[nH]/c(=N/C(C)C)/[nH]c(n1)OC
211.2642 211.2642 211.143311 211.1428815 2.03436459 comp
11 11 232.016676 C1H5O9N5 231.0093994 7.780558586 1
DetailTable: 1 Items
CSID Name Formula SMILES MW AvgMass MonoisotopicMass SearchMass DeltaPPM Adduct
3008 Diuron C_{9}H_{10}Cl_{2}N_{2}O CN(C)/C(=N\c1ccc(c(c1)Cl)Cl)/O
233.0945 233.0945 232.017014 232.016676 1.45673122 comp
12 12 234.1254712 233.1181946 5.222514153 2
DetailTable: 2 Items
CSID Name Formula SMILES MW AvgMass MonoisotopicMass SearchMass DeltaPPM Adduct
4E+06 Stiripentol C_{14}H_{18}O_{3} CC(C)(C)C(/C=C/c1ccc2c(c1)OCO2)O
234.29092 234.2909 234.125595 234.1254712 0.528861389 comp
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Summary and Questions
The HRAM performance has better sensitivity than LC-MS/MS for EPA 539 with additional confirmation such as product ions and a spectral library match.
Further the same injection can be used for identification of non-target and unknowns simultaneously, using Sieve software and/or looking for formation and degradation products using Compound discovery software.
Dr. Andrew Eaton (andyeaton@eurofinsus.com)Ali W. Haghani (alihaghani@eurofinsus.com)
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