development of the world’s most sensitive triple quad ms · the world’s most sensitive triple...
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Development ofthe World’s Most
SensitiveTriple Quad MS
LC/MS DivisionAgilent TechnologySanta Clara, CA
Triple Quad? MRM (Multiple Reaction Monitoring) ?
170 210 250 290
210
222
268 280165
Quad Mass Filter (Q3)Quad Mass Filter (Q1)
Collision Cell
Spectrum with backgroundions (from ESI)
Q1 lets onlytarget ion 210 pass through
190 210
210
Collision cell breaks ion 210 apart
150 170 190 210
210158
191
Q3 monitors onlycharacteristic fragments 158 and 191 from ion 210 for quant and qual.
160
158
190
191
no chemical background
Q U A N T I TAT I O N - S E N S I T I V I T Y
“…only about one out of every 103 to 105 ions generated by ESI at atmospheric pressure
is actually detected using present instrument designs.”
(Page, Smith, et al; JASMS, 2007, 18, 1582-1590)
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6490 Triple Quadrupole LC/MS System with iFunnel Technology
N E W 6 4 9 0 T R I P L E Q U A D R U P O L E L C / M S
Zeptomole Sensitivity is here!Much higher performance in a
Much smaller box!iFunnel Technology• A new paradigm for atmospheric ion sampling• 10X sensitivity breakthrough for ESI• Improves assay robustness; simplifies sample prep
Curved, Tapered Collision Cell• Efficient ion transmission; Reduced noise• Compact design (25% smaller)
New scanning capabilities• 0.4 m/z precursor isolation reduces interference• Dynamic MRM• Data dependent scanning
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Agilent Jet Stream
• Thermal confinement of ESI plume
• Efficient desolvation to create gas phase ions
• Creates an ion rich zone
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N E W I F U N N E L T E C H N O L O G Y
Hexabore Capillary
• 6 capillary inlets• Samples 10X more ion
rich gas from the source• Captures the majority of
the gas from the source region
Dual Ion Funnel
• Removes the gas but captures the ions
• Removes neutral noise• Extends turbo pump life
iFunnel Technology captures 10X more ions
Agilent Jet Stream Ion Generation
Gas Dynamics View
The super-heated sheath gas collimates the nebulizer spray and creates a dramatically “brighter source”
Nozzle voltage
Resistive samplingcapillary
Nebulizing gas
Super-heated sheath gas
Heated drying gas
Enhanced efficiency nebulizer
Patent Pending
6
7
SIX bores
HALF as long
Six bores, Half the restriction means…
• Nearly 12x the amount of atmospheric gas sampledAND
• 5 - 10x the number of ions sampled over wide mass range.
But how do we handle all the extra gas molecules?
Compare:Standard
Capillary is180mm
6 Bore Capillary
Agilent’s Hexabore Atmospheric Sampling…. The Most Sensitive…. By Far.
Hexabore Sampling Capillary
• Hexabore sampling capillary increases ion sampling efficiency up to 10X compared to single bore
• Capillary length is reduced by 50% • minimize high mobility ion loss• pulls in more ion rich gas
Ion sampling gains are most dramatic with the combination of both Agilent Jet Stream technology with the hexabore sampling capillary
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High Pressure Stage 1
Low Pressure Stage 2
ExistingRF IonGuide
Agilent’s Two Stage Ion Funnel Can Handle the Gas Load
Stage 18-12 Torr
Stage 21-3 Torr
Stage 1 offset breaks up the high pressure gas exiting the Hexabore Capillary
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Novel Curved and Tapered Collision Cell DesignEfficient Ion Transmission over wide mass rangeReduces transmission of ionizer generated noise
Larger (5 mm)
EntranceAperture
Accepts more Ions
Smaller (3.5mm)
ExitApertureFocusesIons into
MS2
RF & DCVoltages Set
Confinement and Axial Acceleration
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Improved Ion Sampling Efficiencies with iFunnel
Agilent Jet Stream positive ion
200 400 600 800 1000 1200 1400m/z
10x
14x11x
10x7x
113
302 1034
200 400 600 800 1000 1200 1400
1222
922622
322
118
m/z
5x5x
7x
7x7x 6490
6460
Agilent Jet Stream negative ion
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New 6490 Increases Signal but also Reduces Noise
6X gain in signal shown in previous slide translates into 10X gain in signal-to-noise for alprazolam relative to the 6460 as shown below
6460S/N = 1,157:1 where noise = 1xRMS
6490S/N = 11,640:1 where noise = 1xRMS
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1 pg Chloramphenicol (8X) 1 pg Acid red 4 (9X)
6460 BB1: CAT CConly
Triton
Relative Increase insignal of Triton vs. 6460
6 x
3x103
0
1
2
0.5 1 1.5 2Acquisition Time (min)
6460
64903
00.10.20.30.40.50.60.70.80.9
11.1
0.2 0.4 0.6 0.8 1Acquisition Time (min)
6490 with iFunnel vs 6460 in negative ion mode
x103
6490
6460
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AT T O G R A M D E T E C T I O N L I M I T S W I T H T H E 6 4 9 0
100 attograms of verapamilinjected on column
Six orders of linear dynamic range(100 attograms to 100 picograms)
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Unprecedented Six logs of Linearity with 6490
Y = 65283 x – 0.390412R2 = 0.9976
Femtograms
Are
a R
espo
nse
1
10
100
1000
10000
100000
1000000
10000000
0,1 1 10 100 1000 10000 100000
100 Attogram to 100 Picogram varapmil on-column
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New 6490 Triple installation Spec: S/N > 10,000:1 so the 6490 should be able to detect 1 fg ?
1 pg reserpine on-column S/N > 22,605:1
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6490 Triple - Attogram Sensitivity for Reserpine1 fg of reserpine injected on-column
Acquisition Time [min]
S/N scales as expected
NO gaming the noise spec
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Agilent Triple Quads: 2006 – 2010Relentless increase in signal-to-noise specs
0
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2006 2007 2008 2009 2010
64106430
6460
6490SN
R 1
pg
rese
rpin
e
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Res
erpi
ne R
espo
nse
(Sig
nal/N
oise
)
0
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2004 2005 2006 2007 2008 2009 2010
AB/SciexAgilent
New Agilent 6490
Agile
nt 6
460
Agile
nt 6
430
Agile
nt 6
410
T R I P L E Q U A D R U P O L E I N S T R U M E N T S P E C I F I C AT I O N S
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2.5 Femtogram on-columnArea RSD = 23.7% (n=6)Mean accuracy = 104
50 pg/mL Fluticasone in plasma with 6460
Acquisition Time (min)0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4
*1.534 min.1x10
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5
5.1
5.2
5.3
MRM unit / unit resolution (0.7 amu / 0.7 amu)
O
CH3
F
F
OH
H
CH3
OS
F
O
CH3O
CH3
Fluticasone propionate
20
5 pg/mL Fluticasone in plasma with new 64902.5 Femtogram on-column
Area RSD = 8.7% (n=5)Mean accuracy = 104
Acquisition Time [min]
0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5
1x10
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5
5.1
5.2
5.3
1.765 min.
O
CH3
F
F
OH
H
CH3
OS
F
O
CH3O
CH3 MRM enhanced / unit resolution (0.4 amu / 0.7 amu)
Fluticasone propionate 1x10
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5
5.1
5.2
5.3
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1x10
5
6
7
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4
Q1 Unit (0.7 amu)Q2 Unit (0.7 amu)
The Value of Enhanced Resolution on 6490Fluticasone propionate (inhalant) in human plasma 2.5 fg on-column)
Q1 Enhanced (0.4 amu)Q2 Unit (0.7 amu)
1x10
5
6
7
Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4
Enhanced Resolution gives increased selectivity for analytes in complex matrices
22
Agilent Dynamic MRM application kitsOur approach to make you more productive
G1733AA - Pesticide DMRMData Base Kit
• Database with conditions, transitions, and retention times for 600 compounds
• Pre-configured methods for 300 pesticides for different LC configurations
• ZORBAX Eclipse Plus UHPLC column
• Small suite positive and negative ion test mix for method validation and adjustment
• Extensive documentation and free trial SampliQ QuEChERS kits
Multi residue pesticide method Compounds included in method
313 compounds including 9 isomers
Acephat Butocarboxim-sulfoxid Desmedipham Fenamiphos-sulfon Flusilazol Metamitron Oxadiazon Proquinazid Terbutylazin
Acetamiprid Buturon DMSA Fenamiphos-sulfoxid Flutolanil Metazachlor Oxadixyl Prosulfocarb Tetraconazol
Acrinathrin Cadusaphos Dichlofluanid Fenarimol Flutriafol Metconazol Oxamyl Pymetrozin Thiabendazol
Aldicarb Carbaryl Dichlorvos Fenazaquin Foramsulfuron Methabenzthiazuron Oxasulfuron Pyraclostrobin Thiacloprid
Aldicarb-sulfon Carbendazim Diclofop-methyl Fenbuconazol Formetanat-Hydrochlorid Methacrifos Oxydemeton-sulfon Pyrazophos Thiamethoxam
Aldicarb-sulfoxid Carbofuran 3 hydroxy Dicrotophos Fenbutatinoxid Formothion Methamidophos Paclobutrazol Pyridaben Thifensulfuron-methyl
Alloxydim Carbofuran Diethofencarb Fenhexamid Fosthiazat Methidathion Paraoxon-methyl Pyridaphenthion Thiodicarb
Amidosulfuron Carbosulfan Difenoconazol Fenobucarb Fuberidazol Methiocarb Penconazol Pyridat Thiofanox sulfon
Aminopyralid Carfentrazone-ethyl Difenoxuron Fenoxaprop free acid Furathiocarb Mthiocarb-sulfon Pencycuron Pyrifenox Thiofanox sulfoxid
Amitraz Chlorantraniliprol Diflubenzuron Fenoxycarb Halosulfuron-methyl Methiocarb-sulfoxid Pendimethalin Pyrimethanil Thiofanox
Asulam Chlorfenvinphos Diflufenican Fenpiclonil Haloxyfop free acid Methomyl Pethoxamid Pyriproxifen Thiophanat
Atrazin Chlorfluazuron Dimefuron Fenpropimorph Hexaconazol Methoxyfenozid Phenmedipham Pyroxsulam Thiophanat-methyl
Avermectin B1a Metabolite Chloridazon (Pyrazon) Dimethenamid Fenpyroximat Hexaflumuron Metobromuron Phenthoat Quinalphos Tolclophos-methyl
Avermectin B1a Chlorimuron-ethyl Dimethoat Fenthion Hexythiazox Metolachlor Phorat Quinmerac Tolylfluanid
Avermectin B1b Chloroxuron Dimethomorph Fenthion-oxon Imazalil Metosulam Phosalon Quinoxyfen Topramezone
Azimsulfuron Chlorsulfuron Dimoxystrobin Fenthion-oxon-sulfon Imidacloprid Metoxuron Phosmet Quizalfop free acid Tralkoxydim
Azinphos-ethyl Chlozolinat Diniconazol Fenthion-oxon-sulfoxid Indoxacarb Metrafenon Phosmet-oxon Quizalofop-ethyl Triadimefon
Azinphos-methyl Chromafenozid Dioxathion Fenthion-sulfon Ipconazol Metribuzin Phosphamidon Rimsulfuron Triadimenol
Azoxystrobin Clethodim Diuron Fenthion-sulfoxid Iprodion Metsulfuron-methyl Phoxim Rotenone Triasulfuron
Beflubutamid Clofentezin EPN Flazasulfuron Iprovalicarb Mevinphos Picoxystrobin Sethoxydim Triazofos
Benalaxyl Clomazon Epoxyconazol Flonicamid Isoproturon Molinat Pinoxaden Siduron Tribenuron-methyl
Benfuracarb Clopyralid Ethaboxam Florasulam Isoxaflutol Monocrotophos Piperonyl butoxid Silthiopham Trichlorfon
Bensulfuron-methyl Clothianidin Ethiofencarb Fluazifop free acid Kresoxim methyl Monolinuron Pirimicarb Simeconazol Tricyclazol
Benthiavalicarb Cyazofamid Ethiofencarb-sulfon Fluazifop-P-butyl Lenacil Monuron Pirimiphos-methyl Spinosad Trifloxystrobin
Bifenazat Cycloxydim Ethiofencarb-sulfoxid Flufenacet Linuron Myclobutanil Pirmicarb-desmethyl Spirotetramat Triflumizol
Bifenox Cyhexatin Ethion Flufenoxuron Lufenuron Napropamid Prochloraz Spiroxamin Triflumuron
Bispyribac Cymoxanil Ethiprol Flumetsulam Malaoxon Neburon Profenophos Sulfosulfuron Triflusulfuron-methyl
Bitertanol Cyproconazol Ethirimol Fluometuron Mandipropamid Nicosulfuron Promecarb Tebuconazol Triforin
Bromacil Cyprodinil Ethofumesat Fluopicolid Mecarbam Nitenpyram Propamocarb Tebufenozid Trinexapac-ethyl
Bromuconazol Cyromazin Ethoprophos Fluoroglycofen-ethyl Mepanipyrim Novaluron Propaquizafop Tebufenpyrad Trit iconazol
Bupirimat Daminozid Etofenprox Fluoxastrobin (E) Mesosulfuron-methyl Nuarimol Propargite Teflubenzuron Tritosulfuron
Buprofezin DEET Famoxadon Fluquinconazol Mesotrione Ofurace Propiconazol Tembotrion Zoxamid
Butocarboxim Demeton-S-methyl Fenamidon Fluroxypyr Metaflumizone Omethoat Propoxur Tepraloxydim
Butoxycarboxim Demeton-S-methyl-sulfon Fenamiphos Flurtamon Metalaxyl Orthosulfamuron Propyzamid Terbutryn
Pesticide MRM Data Base G1733AA
Page 25
Multi residue pesticide method Comparison of MRM and Dynamic MRM
Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Compounds (10msec / Compound
Cycle Time (msec)
Max Coincident
Cycle Time (msec)
MRM
50 80
400 400 400 40020 40 40
Dynamic MRM
500 800 1000
30
Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4
700100 70
• 2 x shorter cycle times supports narrow chromatographic peaks, more analytes or longer dwell per analyte.
Multi residue pesticide method Dynamic MRM simulation
Time >>>
Abu
ndan
ce>>
>
ConcurrentCompounds
Multi residue pesticide method MRM traces for tomato extract spiked at 10 µg/kg
5x10
0
0.1
0.2
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+ESI MRM Frag=380.0V [email protected] (116.0000 -> 70.1000) tomato 5 pg_uL.d
Counts vs. Acquisition Time (min)7.6 7.7 7.8 7.9 8 8.1 8.2 8.3 8.4 8.5
more than 160 MRMtransitions in 1 min
Dynamic MRM viewer – display of concurrent MRMsQuantitative analysis of pesticides (dMRM)
Page 29
Compounds at a glance – peak review in MH Quant
Results for black teaMRM traces for black tea extract spiked at 0.5 pg/µl
nearly all compounds were found
equates to 2.5 µg/kg in the sample 25% of the maximum residue limit
Results for black teaCoverage of method for black tea
0
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350
0,5 2,5 5 25 50 250 500
262298 302 312 312 312 312
concentration in µg/kg in black tea
number of compounds dectected in spiked black tea
All compounds found in black tea
MRL
Results for black teaDimethoate/Omethoate (No. 1 in “Check Your Scope” ranking)
0.5 µg/kg 2.5 µg/kg 5 µg/kg 25 µg/kg 50 µg/kg
Omethoate
Dimethoate
Results for black teaOxamyl and Carbendazim (High in “Check Your Scope” ranking)
0.5 µg/kg 2.5 µg/kg 5 µg/kg 25 µg/kg 50 µg/kg
Oxamyl
Carbendazim
Results for tomato extractCoverage of method for tomato matrix
0
50
100
150
200
250
300
350
0,1 0,5 1 5 10 50 100
281308 309 313 313 313 313
concentration in µg/kg in tomato
number of compounds dectected in spiked tomato
All compounds found
MRL
Ultra Trace Levels of Pesticides in Potable Water
Simetryn S/N = 9
Atraton S/N =: 819
Simazine S/N = 18
Secbumeton S/N = 51
Prometon S/N = 40
Atrazine S/N = 28
Triazine herbicides analyzed at 50 parts per quadrillion
6490 Triple Quad with on-line enrichment
36
Triggered MRM – What Problem Does tMRM Solve?
Gap in MRM trace, numerically filled in.Typically > 100 msec
Data dependent product ion scans leaves gaps in MRM• mix of raw and synthetic points• higher RSDs and higher LOQs
DDA –scan switch to PIS
Targeted Quant + Compound Identification
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Triggered MRM data acquisitionNew acquisition mode for enhanced confirmation• Triggered MRM is new data dependant acquisition mode for the
confirmation of target compounds• When the signal heights of the primary MRMs exceed a given threshold up
to 9 additional MRM transitions are triggered• Triggered MRM makes use of the well established Dynamic MRM
functionality• Triggered MRM has advantages over data dependant product ion scans
because:• It is more sensitive due to longer dwell times per transition and due to the ideal
collision energies for each transition• Peak shapes of the primary transitions are not compromised due to constant cycle
times• In spectra fragment ratios are very constant due to relatively long dwell times per
transition• Triggered MRM spectra can be saved in an user defined library and can
be searched from the qualitative and quantitative software.
tMRM for up to 8 Confirmatory Ion Transitions- when primary compound MRM triggers confirmatory MRMs
Plot counts versus m/z for product ions “Composite MS/MS spectra for compound b”
coun
ts
100 200 300
Product %MRMIon Scan Sensitivity
TimeQTRAP
5500 ~ 100 msec ~ 10%
Vantage ~ 150 msec ~ 2%Xevo ~ 150 msec ~ 2%
6490 ~ 30 msec 100%
Faster compound ID at higher sensitivity with tMRM
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Triggered MRM data acquisitionMethod parameters
1. Primary and secondary MRM transitions are imported from Optimizer database (in total up to 10 MRMs per compound)
2. Thresholds for triggering are specified based on response of primary transitions at LOQ3. Number of repeats for triggering is specified – typically 5 repeats are sufficient for
reproducible MRM spectra4. Cycle time stays constant to allow for reproducible quantitation whereas dwell times are
maximized based on number of concurrent MRMs
1.2.
3.4.
Quantitative analysis of pesticidesUser editable Triggered MRM spectra library for pesticides
Quantitative analysis of pesticidesAutomatic creation of triggered MRM library from MH Quant
Quantitative analysis of pesticidesTriggered MRM traces of tebufenpyrad in QC sample (50 ppb)
2x10
0
0.2
0.4
0.6
0.8
1
Cpd 1: Tebufenpyrad: +ESI MRM:361 (9.611-9.752 min, 15 Scans) Frag=165.0V [email protected] (334.2…
147.1000
117.0000
132.100091.1000 105.1000
2x10
-1
-0.5
0
0.5
1
Cpd 1: Tebufenpyrad: +ESI MRM:361 (9.611-9.752 min, 15 Scans) Frag=165.0V [email protected] (334.2…
147.1000
117.0000132.100091.1000 105.1000
2x10
0
0.2
0.4
0.6
0.8
1
Tebufenpyrad C18H24ClN3O + MRM CAS_Library.mslibrary.xml
117.0000 145.0000
132.100091.1000 105.1000
Counts vs. Mass-to-Charge (m/z)75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170
Library match score: 96.75
Quantitative analysis of pesticidesLibrary match for tebufenpyrad in QC sample
Acquired TMRM spectrum
TMRM library spectrum
Comparison Acquired vs.TMRM library spectrum
Quantitative analysis of pesticidesLibrary match for matrix compound in ginger extract
Acquired TMRM spectrum
TMRM library spectrum
Comparison Acquired vs.TMRM library spectrum
Quantitative analysis of pesticidesBatch-at-a-glance – quantitation of tebuthiuron in chamomile extract
Signal for tebuthiuron for quantifier and qualifier- RT slightly higher than reference RT but within accepted RT range- Qualifier ratio higher than expected (183% of expected value)
Quantitative analysis of pesticidesTriggered MRM traces of tebuthiuron in QC sample (50 ppb)
2x10
0
0.25
0.5
0.75
1
Cpd 14: Tebuthiuron: +ESI MRM:370 (6.070-6.250 min, 19 Scans) Frag=105.0V [email protected] (229.10…
172.1000
62.000089.1000
74.0000 116.0000157.1000
2x10
-1
-0.5
0
0.5
1
Cpd 14: Tebuthiuron: +ESI MRM:370 (6.070-6.250 min, 19 Scans) Frag=105.0V [email protected] (229.10…
172.1000
62.0000 89.100074.0000 116.0000 157.1000
2x10
0
0.25
0.5
0.75
1
Tebuthiuron C9H16N4OS + MRM CAS_Library.mslibrary.xml
172.1000
62.0000 116.000089.100074.0000 157.1000
Counts vs. Mass-to-Charge (m/z)60 70 80 90 100 110 120 130 140 150 160 170
Library match score: 97.93
Quantitative analysis of pesticidesLibrary match for tebuthiuron in spiked chamomile extract
Quantitative analysis of pesticidesLibrary match for matrix compound in spiked chamomile extract
200 zeptomoles
verapamilInjected on-column
Acquisition Time (min)
+ MRM (455.3 -> 164.9)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1x10
4.2
4.3
4.4
4.5
6490 Achieves Zeptomolar Detection Limits
50
The 6490 has Zeptomole Capabilitywhat is a zeptomole?
Photo of new 6490 taken from 1 meter distanceHow far away is 1021 meters?
51
The Milky Way Galaxy as seen from Hubble1021 meters is at the edge of the known universe
52