Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
Making LC/MS Analyses Accessible to
Non Specialists
Tony Brand, Ph.D.Applications ScientistAgilent Technologies
November 17, 2010
Making LC/MS Analysis Accessible1
Topics for Today
WorkflowsEnabling Technologies
Small particle LC columnsVersatile LC systems to take advantage of LC columnsSimple robust LC/MS instruments
Single QuadrupoleTime of Flight(QQQ, QTOF, Ion Trap)
The Multi-mode Source and Agilent JetStream TechnologyWhat should an end user need to know?
Reporting MS data or “just give me the answer”?Enabling Software
November 17, 2010
Making LC/MS Analysis Accessible2
The Target User
v Users who need a relatively simple answer from an LC/MS instrument with minimal interaction.
v Users who primarily don’t have time to manage the LC/MS instrument. They want the instrument to wake up, run samples and go to sleep with miminal consumable waste. They want the instrument to maintain itself in a state of readiness.state of readiness.
v Environments where there are limited LC/MS experts, but large numbers of users!
November 17, 2010
Making LC/MS Analysis Accessible3
Workflows – What’s the question?
Did I make what I thought I made?
Synthetic Chemists
Medicinal Chemists
What’s in this stuff?
What is it contaminated with?
Environmental and Toxicological Screening
Raw materials monitoring
November 17, 2010
Making LC/MS Analysis Accessible4
The Theory of Common LC/MS Ionization Modes
+ ++
++ +
+
++
+-
--
--
++++--
Charged Droplets
Atmospheric PressureElectrospray Ionization (ESI)
Atmospheric Pressure Chemical Ionization (APCI)
Analyte containing aerosol
Vapor
Heat
Corona discharge+ ++
++
++
++
+----
-+
+++++--
+
+++++-- +
Analyte IonsHeat
Coulombic Explosions
++ +
+
++
+ ++
++
+
+Charged reagent gas
Corona discharge
Ø Spray into a charged environmentØ Droplets evaporate and explodeØ Single ion droplets evaporate
Ø Spray into a heated environmentØ Vaporize into the gas phaseØ Corona discharge creates ionsØ Charge transfer generates analyte ions
November 17, 2010
Making LC/MS Analysis Accessible5
Enabling Technologies
From HPLC to RRLC on the 1220 Infinity LCVarying Pore Size and Column Length
Flow rate: 1.2 mL/min at 40°C
From HPLC to RRLC
From HPLC to RRLC
From HPLC to RRLC
From HPLC to RRLC
November 17, 2010
Making LC/MS Analysis Accessible7
Phenomenal Column Technologies: Identical Interchangeable ChemistriesTotally Porous 1.8µm particles and Superficially Porous 2.7µm particles
Pmax= 375bar4.6 min!
code
ine
oxyc
odon
e
MD
Aamph
etam
ine
hydr
ocod
one
met
ham
phet
amin
eM
DM
A
phen
term
ine
stry
chni
neM
DE
A
hero
in
coca
ine
mep
erid
ine
traz
odon
e
PC
P
oxaz
epa
m TH
C
diaz
epam
proa
dife
n
tem
azep
am
alpr
azol
amm
etha
done
nitr
azep
am
vera
pam
illo
raze
pam
Column: RRHT Eclipse Plus C18, 2.1 x 100mm, 1.8um
Pmax= 280bar
met
ham
phet
amin
e
coca
ine
traz
odon
e
met
hado
ne
vera
pam
ilColumn: Poroshell 120, 2.1 x 100mm, 2.7um
Agilent LC/MS Toxicology Test Mixture with 25 components analyzed on an Agilent 1200 RRLC + Agilent 6410 QQQ.
A: 5mM ammonium formate w/ 0.01% formic acid (1L water + 0.3153g ammonium formate + 0.1mL formic acid), B: acetonitrile w/ 0.01% formic acid (1L acetonitrile + 0.1mL formic acid); 0.5mL/min; 10%B at t0, ramp to 15%B in 0.5min, ramp to 50%B in 2.5min, ramp to 95%B in 1min, hold 95%B for 2min; stop time 6min, post run 2min; Sample: injector program: draw 5µL water, draw 1µL LC/MS Toxicology Test Mixture (PN 5190-0470), inject; TCC=60oC; MS Source: electrospray AP-ESI, drying gas temperature and flow: 350oC, 12L/min, nebulizer gas pressure: 30psi, capillary voltage: 2000V; MS Acquisition: dynamic MRM (+ve ionization polarity)
Pmax= 280bar4.5 min!
code
ine
oxyc
odon
e
MD
Aam
phet
amin
e
hydr
ocod
one m
etha
mph
etam
ine
MD
MA
phen
term
ine,
st
rych
nine
MD
EA
hero
in
coca
ine
mep
erid
ine
traz
odon
e
PC
P
oxaz
epam
, nitr
azep
am
TH
C
diaz
epam
proa
dife
n
tem
azep
am
alpr
azol
amm
etha
done
vera
pam
illo
raze
pam
Column: Poroshell 120, 2.1 x 100mm, 2.7um
November 17, 2010
Making LC/MS Analysis Accessible8
SOLIDCORE
Matching Needs to Technical Capabilities
1260 - Adds fast gradients at up to 600 bar• Total versatility….. All 1260 modules• Add high precision low carryover autosampler options
1290 – Adds very fast gradients at up to 1200 bar§ 1200 bar pressure rating§ High sensitivity Diode Array Detector @ 160Hz§ Highest precision, lowest carryover
• Both high and low pressure pump mixing options• X 10 UV sensitivity • Available in Bio-Inert model• Available as SFC (liquid CO2) system
1220 – Integrated Simplicity• Injection from vials or manual injection• Single wavelength UV detection• 600 bar pressure rating• Isocratic or Low pressure mixing versions
RRLC – Rapid Resolution LCThe ability of a LC-system to make use of sub-two micron particle size columns to achieve faster - more rapid - and higher resolvinganalysis. This is also called UHPLC and has many other synonyms.
November 17, 2010
Making LC/MS Analysis Accessible9
Agilent Jet Stream TechnologyThe sample is nebulized into a charged environment and inside a sheath of superheated nitrogen gas.
ü Narrow chromatographic peak widthsü Very high signal to noiseü Ionization by multiple mechanismsü Matched to the Agilent 1290 Infinity LCü Integrated in the 6150 SQ and 6230 TOF
UV vs MS Peak Width Comparison
2E+5
3E+5
4E+5
150
200
250
300UV Signal (mAU)
UVAJS Glafenine
Labetalol
Chrysin
di-n-pentyl phthalate
1290/6150 system, 1 min Gradient (water/acetonitrile with 0.05% TFA), 2.1x30mm Eclipse Plus C18, 1.8um
0E+0
1E+5
0
50
100
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
UV Signal (mAU)
Minutes
Caffeine
November 17, 2010
Making LC/MS Analysis Accessible11
The Agilent Multi-Mode Source
üSimultaneous ESI and APCIü Ionizes more than ESI or APCI aloneüTime saving
Field-shapingelectrodes
IR emitters
Sensor for vapor temperature
APCI Counter electrode
November 17, 2010
Making LC/MS Analysis Accessible12
Building the Right Solution
External Tray for Standard Autosampler
Vials discarded here on completion (with optional delay)
Up to 17 vials in external traySampler arm moves them inside
November 17, 2010
14
LC and MS options: SQ and TOF instruments
6130B SQ adds Versatility Mass range 2-3000m/zMax scan speed 5250u/S
6150 SQ with Agilent JetStream TechnologyMass range 2-1350m/zMax scan speed 10000u/S50 x more sensitive than 6120B+/- switch time 20msec
12901200 bar Highest precisionHighest speed
1260600 bar Versatility
6120B SQ Simple, robustMass range 2-2000m/zMax scan speed 2500u/S+/- switch time 300msec
Max scan speed 5250u/S10x more sensitive than 6120B+/- switch time 50msec
Versatility
1220600 barSimplicityLowest cost
November 17, 2010
Making LC/MS Analysis Accessible15
Fast Scan POS/NEG Switching at 10,000 Da / sec and 20 msecGives more confident compound identifications
���280 282 284 286 288 2900
20
40
60
80
100
286.
0
287.
0
285.
0 Peak #2, positive ion Isotope ratios
M/Z Theory Obs.286 13.3 11.5287 37.7 35.2288 4.9 4.6289 1.9 2.2
288.
0
289.
0
Theory
Observed
���0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0
200000
400000
600000
800000
1000000
1200000*MSD3 BPC, MS File (D:\6150 DATA\SULFA-CONDPN-01.D) AJS-ES, Pos, Fast Scan
#2 ���280 282 284 286 288 290
282.
9 Peak #2, negative ionIsotope ratios
M/Z Theory Obs.284 13.3 13.6285 37.7 36.0286 4.9 4.1287 1.9 2.3
Theory
Observed
���280 282 284 286 288 2900
20
40
60
80
100
284.
0
285.
0
286.
0
287.
0
Accurate isotope ratios
���0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0
50000
100000
150000
200000
*MSD4 BPC, MS File (D:\6150 DATA\SULFA-CONDPN-01.D) AJS-ES, Neg, Fast Scan
Base Peak Chromatogram
November 17, 2010
Making LC/MS Analysis Accessible16
Agilent 6224 TOFSensitivity 10 pg: 20:1 reserpine.
MS mode, ESI,on-column10:1 RMS (typical 30:1 RMS)
Agilent 6230 TOF with Agilent JetStream TechnologySensitivity 2 pg: 20:1 reserpine.
MS mode, ESI,on-column10:1 RMS (typical 30:1 RMS)
Mass resolution > 20,000 FWHM at m/z 1522 after auto-tune> 10,000 FWHM at m/z 118 after auto-tune
Mass accuracy < 2 ppm RMS (10 repeat injections)Mass range 20000 m/zAcquisition speed 40 scans/sec
10:1 RMS (typical 30:1 RMS)Mass resolution > 20,000 FWHM at m/z 1522 after auto-tune
> 10,000 FWHM at m/z 118 after auto-tuneMass accuracy < 2 ppm RMS (10 repeat injections)Mass range 20000 m/zAcquisition speed 40 scans/sec
November 17, 2010
Making LC/MS Analysis Accessible17
How Much Accuracy is Needed?
Reserpine (C33H40N2O9) has a protonated ion at 609.28066
Single quad reports mass to +/- 0.1 = 165 ppm
Number of possible formulas using only C, H, O & N:
• 165 ppm 209
• 10 ppm 13• 10 ppm 13
• 5 ppm 7
• 3 ppm 4
• 2 ppm 2
Accurate mass reduces number of candidates and risk of investing effort with the wrong molecule
November 17, 2010
Making LC/MS Analysis Accessible18
LC and MS options: SQ and TOF instruments
6130B SQ Adds Versatility
6150 SQAdds Agilent JetStream Technology
12901200 bar Highest precisionHighest speed
1260600 bar Versatility
6230 TOFAdds Agilent JetStream Technology,5x Sensitivity + greater peak capacity
6224 TOF
6120B SQSimple, robust
Versatility
1220600 barSimplicityLowest cost
November 17, 2010
Making LC/MS Analysis Accessible19
6224 TOFSimple, StableSub 1-2ppm mass accuracy
Implementing Solutions
Analysis of fumonisin, FB1 & FB2 Mycotoxins, in CornRef. Syed Salman Lateef, Agilent publication number: 5990-6560EN
Fumonisins are fungal metabolites, which can cause mycotoxicosis when ingested. The FB1, FB2 and FB3 types can contaminate corn samples. Amounts in food and feed are regulated.
“The major challenge … is that different extraction procedures may be required for different food matrices.”
LC/MS: Agilent 1220 Compact LC plus Agilent 6120 Single Quadrupole LC/MS
November 17, 2010
Making LC/MS Analysis Accessible21
ü Simplified user interface for anyone using LC, LC/MS single quad, or LC/MS TOF systems
ü Expandable to large numbers of users and multiple instruments in
Agilent MassHunterEasy-Access Software
Walk-up LC and LC/MS
ü Expandable to large numbers of users and multiple instruments in different locations
ü Configurable to match laboratory SOPs, specific terminology, usage rules, etc.
ü One skilled user required for setup and administration
ü One semi-skilled user can manage multiple systems!
November 17, 2010
Making LC/MS Analysis Accessible22
Sample Login – Step 1 of 3 – Who and How Many?
November 17, 2010
Making LC/MS Analysis Accessible23
Sample Login – Step 2 of 3 – Sample Details
optionalfields
November 17, 2010
Making LC/MS Analysis Accessible24
Sample Login – Step 3 – Confirm and Commit
November 17, 2010
Making LC/MS Analysis Accessible25
Web Access to Instrument Status with Easy Access
Instrument status can be viewed over the internet with a customizable template
November 17, 2010
Making LC/MS Analysis Accessible26
E-mail Notification of System Problems
November 17, 2010
Making LC/MS Analysis Accessible27
Administration – Events and Shutdown Protocol
Automatic calibration of TOF on Wakeup or through Events Scheduler
Automatic calibration of TOF on Wakeup or through Events Scheduler
Events can be scheduled to run at a specific time, every ‘n’ samples or on Wakeup or Startup
Scheduler
November 17, 2010
Making LC/MS Analysis Accessible28
E-mail Notification of Sample Results
Double click to launch Data Browser
Will e-mail ASR files, reports, and/or raw data
November 17, 2010
Making LC/MS Analysis Accessible29
Empirical Formula Confirmation Report (TOF)
Formula input by submitter and system calculates expected monoisotopic mass
Extracted ion chromatogram covering specified adducts
Zoomed spectrum covering Zoomed spectrum covering adduct range
Calculated mass error results
Calculated purity results based on MS, UV or other detector data
November 17, 2010
Making LC/MS Analysis Accessible30
MassHunter Data AnalysisMFE and MFG – Fundamental Algorithms
• Both algorithms are crucial for Agilent’s MassHunter SW• Used in several software applications for accurate mass LC/MS data
• Very competitive and unique compared to other vendor’s offerings
• Molecular Feature Extractor (MFE)• Allows finding molecular features (= compounds) in an LC/MS run inthe MS data domain (identify co-eluting and related mass signals)the MS data domain (identify co-eluting and related mass signals)
• Works on accurate mass MS data from TOF and Q-TOF.
• Molecular Formula Generator (MFG)• Allows determination of molecular formulas of unknown compoundsusing accurate mass TOF MS and Q-TOF MS/MS data
• If multiple adducts or charge states selected, get cross score
• Unique scoring algorithm
November 17, 2010
Making LC/MS Analysis Accessible31
Automated Molecular Feature Extraction - TOF
Report of identified pharmaceutical compounds - Identification of diuretic amiloride with 0.1 ppm.
N
N
Cl
NH2
NH2
N
O
NH2
NH2
November 17, 2010
Making LC/MS Analysis Accessible32
Compound Characterization A good sample
Single peak identified for compound
Utilize similar setup of LC-UV/ELSD/CLND/MS for full evaluation
min*0 0.5 1 1.5 2 2.5 3 3.5
mV
5060708090
*ADC1 A, ELSD (20015\LCM20015-A01.D)
2.5
86
min*0 0.5 1 1.5 2 2.5 3 3.5
mV
20
40
60
80
100
*ADC1 B, CLND (20015\LCM20015-A01.D)
2.5
83
mAU
*DAD1 B, Sig=254,4 Ref=off (20015\LCM20015-A01.D)
2.5
83
for full evaluation
Single MW identified with spectra matching desired compound
min*0 0.5 1 1.5 2 2.5 3 3.5
0
50
100
2.5
83
min0 0.5 1 1.5 2 2.5 3 3.5
0
100000
200000
300000
MSD1 TIC, MS File (C:\CHEM32\1\DATA\20015\LCM20015-A01.D) MM-ES, Pos, Scan, Frag: 150, "TIC" 2
.585
m/z200 300 400 500 600
0
20
40
60
80
100
*MSD1 SPC, time=2.574:2.591 of C:\CHEM32\1\DATA\20015\LCM20015-A01.D MM-ES, Pos, Scan, Frag: 150, "TIC"
Max: 212784
November 17, 2010
Making LC/MS Analysis Accessible33
min*0 0.5 1 1.5 2 2.5 3 3.5
mV
42
44
46
48
50
*ADC1 A, ELSD (20022\LCM20022-E01.D)
2.2
78 2
.352
min*0 0.5 1 1.5 2 2.5 3 3.5
mV
30
35
40
45
*ADC1 B, CLND (20022\LCM20022-E01.D)
1.3
82
1.6
35
1.8
83
2.2
77 2
.348
2.4
46
2.5
49
mAU
60
*DAD1 B, Sig=254,4 Ref=off (20022\LCM20022-E01.D) 1
.636 2
.277
2.3
50
2.4
47
Compound Characterization A poor sample
Clearly several other peaks in system
min*0 0.5 1 1.5 2 2.5 3 3.5
0
20
40
60
1.3
81
1.5
46
1.6
36 1
.695
1.8
85
2.2
21 2
.277
2.3
50
2.4
47 2
.506
2.5
52 2
.604
2.7
35
2.8
37 2
.902
3.0
67
3.1
71 3
.241
min0 0.5 1 1.5 2 2.5 3 3.5
0100002000030000400005000060000
MSD1 TIC, MS File (C:\CHEM32\1\DATA\20022\LCM20022-E01.D) MM-ES, Pos, Scan, Frag: 150, "TIC"
0.1
45 1.3
89
2.2
20 2
.285
2.3
59
2.4
48
2.6
91
2.8
37
m/z150 200 250 300 350 400 450
0
20
40
60
80
100
*MSD1 SPC, time=2.278:2.289 of C:\CHEM32\1\DATA\20022\LCM20022-E01.D MM-ES, Pos, Scan, Frag: 150, "TIC"
Max: 15027 No clean single ion recognizable as the M+H ion from the target mass or formula
November 17, 2010
Making LC/MS Analysis Accessible34
Simple Brief Clear Reports (Single Sample)
ü YES, this really is a single page report from an Agilent SQ LC/MS.
ü Simple clean reports with full options to customize to workflow.
ü Automated e-mailing of PDF reports with ü Automated e-mailing of PDF reports with Easy-Access
ü Includes UV and MS peak purity
ü Reports sample purity for target masses
November 17, 2010
Making LC/MS Analysis Accessible35
The Concise Clean ASR report
November 17, 2010
Making LC/MS Analysis Accessible36
From Data to Information.. Agilent MSD solutions
ASR FileA text file containing
summary data on samples,
chromatograms, peaks and targets
data
Flexible automatable reporting and fast data review at desktop when needed
Analytical StudioReviewerChemStation
Report
ASR ReportBy single sample,batch, or plate
ChemStation
ChemStationRaw Data Folder
MassHunterRaw Data
CS-2-MH translator
MassHunter Qual
MassHunterReporting Engine
November 17, 2010
Making LC/MS Analysis Accessible37
MassHunter Quant
MassHunter Analytical Studio Reviewer (ASR)
A simple data reviewer and reporting tool for LC/MS data
November 17, 2010
Making LC/MS Analysis Accessible38
ASR File Creation Setup: A Single Panel
November 17, 2010
Making LC/MS Analysis Accessible39
Purity – What is it really? The eternal question
Sample purity
The percent area, usually in a UV chromatogram, e.g. 254nm, of a peak determined, usually by mass spec., to contain the target compound.
Peak purity
An evaluation of the spectra, either DAD UV or MS, to look for co-elution. NOT DEFINITIVE, but worthwhile!
November 17, 2010
Making LC/MS Analysis Accessible40
Peak Purity – Diode Array Detector
In ASR, this peak purity data has been distilled down to a single icon or word in a table.
November 17, 2010
Making LC/MS Analysis Accessible41
Peak Purity – MS
November 17, 2010
Making LC/MS Analysis Accessible42
ASR Reporting of Peak Purity
November 17, 2010
Making LC/MS Analysis Accessible43
Conclusions
q Easy-Access manages the LC/MS system to save resources (both people and consumables).
q Easy-Access makes sample submission simple.
q Sub-2 micron particle columns and Poroshell 120 columns make UHPLC practical on low end LC systems.
November 17, 2010
Making LC/MS Analysis Accessible44
q SQ or TOF: what information do you need?
q Analytical Studio Reviewer makes simple elegant LC/MS reporting a reality.
q The rich environments for configuration and customization within the ChemStation and MassHunter platforms offer a unique opportunity to encapsulate LC/MS systems into a simple solution.
Thanks for listening