The Role of Multiple MS pTechnologies in Bioanalytical Analysis

Daniel Pentek*February 3, 2011 -UCONNBioanalytical Chem 395

daniel.pentek@perkinelmer.com

Introduction – Dan Pentek

Education:Wesleyan University: Bachelor’s and Master’s in ChemistryUCONN: Masters in Business AdministrationUCONN: Masters in Business Administration

Work Experience:Yale University Chemical Instrumentation Center (‘83-’93)

Mass SpectrometristPerkin-Elmer / Sciex Joint Venture (‘93-’99)

LCMS InstructorP d t S i li t (d li ti t )Product Specialist (demos, applications, etc.)Technology Sales ManagerApplication/ Demo/ Training Lab Manager

Bayer Healthcare – Pharmaceuticals (‘99-’04)Bayer Healthcare – Pharmaceuticals ( 99- 04)Sr ScientistPrinciple Scientist – Automation and New Technology

PerkinElmer, Inc (‘04-’present), ( p )MS Technology Manager, Product Owner, Product Manager, etc.

2

Topics to be covered:I t d ti Introduction Bioanalytical OverviewMS MS -

MS Market & TechnologiesBasic ionization methods

El t APCI APPIElectrospray, APCI, APPIInterfaces

Orifice, capillary

MS AnalyzersPerformance CriteriaStrengths & weaknesses for bioanalytical analyses

Quadrupole (single &triple quads)Ion traps (3-D and 2-D)Time of flight (linear, reflectron, MALDI)Hybrids (Quad TOF) and othersHybrids (Quad-TOF) and others…FTMS (ICRs and orbitraps)

Block Diagram - Bioanalytical Analysis

Sample PreparationSam

Sample Separation

mple

SPEED i th d i !!Sample Separation

Th

SPEED is the driver!!

Eliminate bottlenecks!

Sample Analysisroug

MS provided a paradigm shift in these areas

Data Analysis

ghpu

4

yt

Separation Based on Mass Has Been Around a Long Time…

Winnowing is a mass separation technique.

5 Slide from Dr. Athula Attygalle

Modern Grain Mill – Separation Based on Mass

A modern grain mill does this more efficiently. In fact, each process taking place inside a

i ill b dgrain mill can be compared to those taking place inside a

mass spectrometer

6 Slide from Dr. Athula Attygalle

What is a (Modern) Mass Spectrometer?

An instrument that separates molecules or atoms (e.g., ICP/MS) based on their mass/charge ratio (m/z).

m is the massΙzΙ is the absolute value of the number of charges on it.

Mass is reported in u mass units or the unified atomic mass unit which is Mass is reported in u, mass units, or the unified atomic mass unit which is defined as 1/12th the mass of carbon 12 (12C) or Da (Daltons) which is more prevalent in the biochemistry world.

Prior to 1993 mass was reported in atomic mass units (amu) which was defined Prior to 1993, mass was reported in atomic mass units (amu) which was defined as 1/16th the mass of oxygen 16 (16O). Masses are still frequently reported in amu, but this is incorrect!

A MS id 2 di i f i f tiAn MS provides 2 dimensions of information:A signal that “something is there” (a signal intensity)The mass of what is there, to a nominal or very precise level of accuracy

LC/MS Historical Perspective

Industry was desperate for a decent, rugged LC/MS interface.

GC/MS required derivatization etcGC/MS required derivatization, etc.Not applicable to most biomolecules (MW, etc.)

Atmospheric pressure ionization (API) ionization along withAtmospheric pressure ionization (API) ionization along withnew interface designs provided the solution.Now, all interfaces are differentially pumped.Now, all interfaces are differentially pumped.

Pumping of interfaces was criticalOrifice – skimmer (nozzle) designsH t d ( ld) ill d iHeated (or cold) capillary designs

LC/MS is a big business now!

Analytical Instruments Technology Segments…

SDI data on analytical instrument technology shows mass spectrometry is a $2.6B market in 2010, and predicts…

…that it will have fastest growth rate (8.6%) of any analytical instrument technology through 2014technology through 2014

*SDI Global 11th Ed. Sept. 2010

Distribution of Mass Spectrometry Techniques**

Avg. CGR >10% Growth Opportunities

$255MCGR 6.2%

$101MCGR 2.3%

$643M

CGR 9.5%$312M

CGR 9.4%$139M

CGR 17.0%$136M

CGR 9.7%$391M

CGR 4.9%

$241M$61M

*SDI Global 9th Ed. Sept. 2006

PKI has product offerings CGR 7.2%CGR 3.8%

LC/MS(/MS): A Marriage of Liquid Chromatography and Mass Spectrometry

Marriage (like any other close relationship) requires: COMPROMISE!COMPROMISE!

LC Person: “MS is just another detector”MS Person: “LC is just an inlet”

What’s good for LC may not be good for MS and vice versa.LC d l ti b f th fi d t h t i t f it LC was around a long time before they figured out how to interface it to an MS.

LC/MS Instrument Block Diagram

LC

Ion Source Interface Mass Analyzers Detectors

-ESI -Orifice – Sk -Quadrupole -CEM

Eluant

-APCI-APPI

-Capillary(Hot or cold)

p-Ion Trap (4 types)-Time of Flight

-Discrete dynode-CCDPhotomultiplier-Photomultiplier

MS System – under vacuumMS System under vacuum

Today’s LC/MS Ionization Methods: All Done at Atmospheric Pressure

On-line techniques:Electrospray (ESI) - Fenn @ Yale ~1984

Shared Nobel Prize in 2002 for this work with K. Tanaka (MALDI) and K. Wüthrich (NMR)Atmospheric Pressure Chemical Ionization (APCI)

Irabarne & Thomson ~1979At h i P Ph t I i ti (APPI)Atmospheric Pressure Photo Ionization (APPI)

Emerging, not as widely used yet.All of the above are done at atmospheric pressureSi ifi t h f t diti l i i ti th d hi h ll d ithiSignificant change from traditional ionization methods which were all done withinthe vacuum chamber.

Off-line techniques:Matrix Assisted Laser Desorption Ionization (MALDI)Desorption Electrospray (DESI)DART- Direct analysis in real timeand more…

PerkinElmer Acquires Analytica of Branford

In ~June of 2009, PKI acquired Analytica of Branford, Inc. (AoB)AoB was company started by John Fenn (share Nobel Prize 2002) and Craig Whitehouse (who built first electrospray source on an LCMS)( p y )

John Fenn Craig Whitehouse

(N b l P i L t 2002)

Company held exclusive IP on electrospray and multiple charging of moleculesIn 1987 AoB started designing ESI sources for many vendors

(Nobel Prize Lecture 2002)

Developed early ESI-TOF MSDeveloped over 75 patents in the field of MS

Ion source technologyIon guide technologyCoupling ESI to o-TOF MS

Significant Overlap in Ionization Techniquesht

1000 kDa

ar W

eigh

100 kDaProteins

Mol

ecul

a

10 kDaPeptides

Electrospray

M

1 kDa

APCIAPPI

Carbamates

APCISteroids

Phenyl ureas

PAHs100 Da

15 PolarityLow High

Electrospray- Spraying a Very (Charged) Fine Mist

High pressure nebulizer gas provides pneumatic assistance

16

High pressure nebulizer gas provides pneumatic assistance…

Electrospray: Ion Evaporation Process for LC/MSDesolvation Process and Ionization Mechanism

• Rayleigh limit is the maximum charge a droplet can hold while maintaining its volume

Evaporation Rayleigh  Coulombic 

maintaining its volume

Ions EjectedEvaporationLimit 

ReachedExplosions

+ + ++ ++ ++ + ++

++ +--

Ions Ejected

+ ++

+++ +

+

+++

++

++ -

--

-

--

++

++ +

+ ++

+

+ +

---

--

++

+++- -- -

++

++

+

++

+---

-

- -

Droplet CapillaryHow well this works depends on the mobile phase composition and flow rate. Must have proper probe positioning, CCDG flow and CCDG temperature and

nebulizer pressure to insure proper ionization of analyte ion

17

nebulizer pressure to insure proper ionization of analyte ion .

Electrospray: Ion Evaporation Process for LC/MSElectric field lines

-

-Electric field lines

(drawing (+) ions in

toward (-) charge) Heated drying gas

++

+

+

+ ++--

-

++

+++

+ -

-

-

-

+ ++ ++-

- -++

++

+--

-- -

+++

+

-- -

+++

+

---

-

+ ++++

--

-++++ + +- -

- -+ +++

+ +-- +++++

+

++ +

+ +

+

+--

- +

-

-

- 6000V at

MS entrance

(under vacuum)

1. Droplet from sprayer with (+) and (-) charges on it

2. Droplet shrinks with mobile phase evaporation

3. Coulombic repulsions rupture droplet ejecting ions (with solvent molecules)

18

(under vacuum)4. Ions of desired polarity follow field lines to capillary tip while being strippedof residual solvent by heated drying gas and they are sucked into the MS entrance

Electrospray

ESI is most widely used LCMS ionization techniqueElectrospray is a concentration dependent technique.

Ease of use and LC flow rates drove source development.Started out as very low flow technique, which wasn’t very compatible with LC.

LC Person: “Use lower flows and narrower column”.MS Person: “Buy ESI probe that has higher gas flows and desolvates better”.

Nanospray takes ESI to much lower flow ratesPreserves precious sampleAllows multiple MS experiments to be run over time (which takes time)

Example of Ion Source Designed for High LC Flow (AB-Sciex).

Bioana

Electrospray- Tips

ModifiersOrganic acids (e.g. formic, acetic) promote ionization of basic compounds (sp3

N t i i )N- containing)Neutral compounds containing nucleophilic lone pairs (sp2 N, sp3 O) can be desorbed by cationization with alkali metal or ammonium ions.Ammonium formate or acetate are recommended buffers ( 2-10 mM optimum, can see suppression effects over 20 mM, 50 mM max.)Salts can interfere with ionization and can cluster to complicate spectrum (but

l id i id tifi ti )also aid in identification)Strong bases or quaternary amines can interfere with positive mode analytesSulfonic acids interfere with negative mode analytesAVOID PHOSPHATE BUFFERS as much as possible

Contaminate ion pathSuppress ionizationpp

Atmospheric Pressure Chemical Ionization (APCI)

Field Free APCI Source Conventional APCI Source

Configuration

Nebulizer

Sample Inlet

Nebulization Gas Inlet

AuxiliaryG I l t

Nebulizer

Sample Inlet

Nebulization Gas Inlet

AuxiliaryG I l t

Nebulizer

Sample InletNebulization

Gas Inlet

AuxiliaryG I l t

Nebulizer

Sample InletNebulization

Gas Inlet

AuxiliaryG I l tGas Inlet

Gas Curtain

Gas Inlet

Gas Curtain

Heater

Gas Inlet

Heater

Gas Inlet

HeaterHot N2

To MSAnalyzer

ElectrodeHeaterHot N2

To MSAnalyzer

Electrode

CoronaNeedle

Hot N2

T MS

Gas CurtainElectrode

CoronaNeedle

Hot N2

T MS

Gas CurtainElectrode

CoronaNeedle

y

SamplingCapillaryCorona

Needle

y

SamplingCapillary

To MSAnalyzer

SamplingCapillary

To MSAnalyzer

SamplingCapillary

Increased analyte concentration around corona needle.

CONFIDENTIAL AND PROPRIETARY INFORMATION OF PERKINELMER, INC © 2009 Perkin Elmer

Increased analyte concentration around corona needle. Decoupling of Ion production from Ion Transport Field.

APCI Reactions

N2 + e- N2+. + 2e-

N2+. + H2O H2O+. + N2

H2O+. + H2O H3O+ + OH.

H3O+ + M [M+H]+ + H2OH3O M [M H] H2O

APCI- Summary

HN is a high flow (0.5-2.0 mL/min.) inlet

Suitable for polar, thermally stable cmpds

Usually, MW < 1000 amu

Probe is heated to facilitate vaporization

Requires nebulizing and auxiliary gas

Requires corona discharge needle to produce ionization (APCI)

Atmospheric Pressure Photo Ionization (APPI)

Emerging technique (about 5 years old)Uses typical 10 eV UV lamp (similar to photo-ionization lamps for yp p ( p pGC). Similar to APCI, but applicable to broader range of compounds.

LC/MS Instrument Block Diagram

LC

Ion Source Interface Mass Analyzers Detectors

-ESI -Orifice – Sk -Quadrupole -CEM

Eluant

-APCI-APPI

-Capillary(Hot or cold)

p-Ion Trap (4 types)-Time of Flight

-Discrete dynode-CCDPhotomultiplier-Photomultiplier

MS System – under vacuumMS System under vacuum

Example: Orifice-Skimmer Interface (AB-Sciex)

Nitrogen GasCurtain Interface

Orifice Plate

DifferentiallyPumped Interface

E V

Orifice Plate

QØSO

UR

CE V

AC

UU

M+

Skimmer

urt ai

nG a

sFlo

w

PI I

ON

S

M C

HA

M

CuA

MB

ER

Bioana

Flexar SQ 300 MS Detector Ion Optics and Vacuum System

Heated Countercurrent

Drying Gas (N2)

Dielectric Capillary

4 stage pumping system: 30/200/200 cartridge turbo with E2M28 rough pump

Patented ion guide technology traversing multiple vacuum stagesPatented ion guide technology traversing multiple vacuum stages

Vacuum Interface: “Protective” Gas & Differentially Pumped Interfaces

Regardless of interface used;UHP nitrogen counter current drying (CCDG) gas keeps non ionized species out of the analyzer regionnon-ionized species out of the analyzer regionCCDG aids in ion de-clustering (with CID potentials)Two stage transition from atmosphere to low pressure region Two stage transition from atmosphere to low pressure region of analyzer (1 x 10-5 torr) CCDG and ions are drawn in due to;;

Pressure differential (both ions & CG)Electric field gradients (ions only)

Interface exit into vacuum offers sample “fragmentation” opportunity

Poor man’s MS/MS ☺Poor man s MS/MS…☺

1st Vacuum Region- Supersonic Expansion Occurs (Mach Disk)

Within a Mach disk, all ions and gas molecules moving in same direction

It should project into the skimmer zone for ion transmission

CID induced by voltage difference between cap. exit and SK

It should project into the skimmer zone for ion transmission

Capillary ID, skimmer ID and pumping speed are all variables requiring optimization

LC/MS Instrument Block Diagram

LC

Ion Source Interface Mass Analyzers Detectors

-ESI -Orifice – Sk -Quadrupole -CEM

Eluant

-APCI-APPI

-Capillary(Hot or cold)

p-Time of Flight

(MALDI- TOF)

Ion Trap (4 types)

-Discrete dynode-CCDPhotomultiplier-Ion Trap (4 types) -Photomultiplier

MS System – under vacuumMS System under vacuum

Analyzer (and System) Criteria

Analyzer Considerations:Mass accuracyResolutionResolutionSensitivityScan speedDynamic range

System Considerations:“Sensitivity”Sample thru-put – Fast LC?Primary application: quantitation qualitative orPrimary application: quantitation, qualitative or…Software- application basedEase of use!!!P iPrice…

Single MS vs.MS/MS Systems:

Single MS systems (quadrupole or TOF)Have the added dimension of mass vs. UV or diode array detectors.However, chemical noise is the limiting factor for sensitivity (S/N) and dynamic range.

So sample preparation becomes a bigger factor!

“MS/MS Systems” Many variations now…

“Triple Quads” - 2 quadrupole MS’s separated by a collision cellTriple Quads - 2 quadrupole MS s, separated by a collision cell“QTOFs” - Quadrupole front end, collision cell, TOF back end“MSn” analyzers- ion traps (3D and 2D) and FTMS’sTOF-TOF separated by collision cellTOF-TOF, separated by collision cellIT – TOF (Shimadzu)

Purposes essentially the same (regardless of analyzer type), select one ion from all the others fragment it and study the fragment ionsfrom all the others, fragment it, and study the fragment ionsMS/MS systems ARE the paradigm shift in bioanalytical analysis

Mass Accuracy- Mass Analyzer Comparison (LC/MS)

Cost: 0 1 2 ($M)

m/z = 1000.00000Cost: ~ 0.1 2 ($M)

m/z 1000.00000 Quadrupole50-200 ppm50 200 ppm

Ion Trap10-100 ppm

FTMS-ICR0.05-1 ppm

Time of Flight1-20 ppm

FTMS –IT (Orbitrap)0.5-2 ppm

Resolution- What is it?

Ability to separate (resolve) adjacent ionsTypically defined as: M/∆MTypically defined as: M/∆M

M: Mass ∆M: Full Width at Half Max.

Quadrupoles: scan at constant peak widthQuadrupoles: scan at constant peak width30/1=30, 300/1=300, 3000/1=3000…

Resolution increases as you go to higher massy g g

TOFs: scan at constant resolution10k res: m/z 10.001, 100.01, 1000.1, 10,001, , , ,

Peak width increases as you go to higher mass

Analyzer Types: Quadrupoles (and hexapoles, octopoles, etc.)

Fundamental parts of virtually all LC/MS systemsServe one of two purposes:Serve one of two purposes:

Ion transmission devices (quad, hex, oct…)Capture and transmit ions from one place to another…

Ion filtering devices (quadrupoles only…)Act as a mass filter (analogous to a magnetic)

Quadrupole Theory

Quad. as a mass filterSeparates ions based on m/z ratioVoltages applied determine “width” of mass window

Quad. made of 4 rods“A pole” - vertical rods; “B pole” – y

L

horiz. rods (by convention)

DC, RF volt. imposed:ro r

++

x

U=(DC)A-(DC)B (FDC) V: RF volt. peak-peak (RFp-p)

2 2

z

V = 7.22 * M * r02 * f 2 ; i.e., V ~ M

‘Time-of-Flight’ (TOF) Analyzers

TOF AnalyzerLinear ModeLinear ModeReflectron Mode

Common Ionization Methods for TOF MSCommon Ionization Methods for TOF MSMALDI ESI

TOF Ion Optics (Linear)

t = time taken to reach the detector (seconds)

m = mass of the ion (kg)

z = number of charges on the ion. If singly charged z = 1charged z = 1

e = magnitude of the charge of an electron (coulombs)

V = Accelerating voltage (volts)

39

V Accelerating voltage (volts)

d = distance an ion travels (flight tube) (meters)Limitations: Kinetic energy distribution

Basic API TOF MS Ion Flight Path

Detector

Pulsing RegionPulsing Region

Horizontal TOF Drift TubeHexapole Ion Guide

Dielectric CapillaryIon Mirror

Grounded API Source

40

Grounded API Source

CONFIDENTIAL AND PROPRIETARY INFORMATION OF PERKINELMER, INC © 2009 Perkin Elmer

Trap-Pulse Mode, Small Packet of Ions

High m/z0V

Bias +30V+10V

Ions filling ion guide, storing ions from last pulse,ion guide exit high Each ion packet smaller than usable pulsing region, mini TOF

0V

Bias +30V+10V

g

Low m/z0V

0V Over fill regionions lost

0V

0V

High m/z

Low m/z

0V

Bias -30V+10V

Initial ion release from ion guide, ion guide exit low

Pulser voltage applied, ions accelerated for TOF

Exclusion regionions lost

Low m/z

High m/z

Low m/z

0V

0V +400V

+200V

Bias +30V+10V

Short pulse of ions from ion guide, ion guide exit high, start storing for next pulse

High m/z

Low m/z

0V

0V

0VBias +30V+10V

start storing for next pulse

Usable pulsing region

41

0V

Slide #: 3014

CONFIDENTIAL AND PROPRIETARY INFORMATION OF PERKINELMER, INC © 2009 Perkin Elmer

MALDI Mass Spectrometry

MALDI = Matrix-Assisted Laser Desorption / Ionization

cas

sS

pec

Ma

MALDI-TOF Ex. Data

Sample Applications (TOF MS)

Biomarker Discovery

MS/MS Systems: Triple Quadrupole

ORIQ1

RNG

Q0 ST RO1

IQ2

ST3 RO3

DF

CEM

6 mTorr1 Torr

RO2 (LINAC)

DF

Varian 550S25B Pump

Leybold 361Backed by D10E

Triple Quad Scanning Modes: Multiple reaction Monitoring (MRM)

TQ’s remain the “Gold Standard” for quantitation because of TQ s remain the Gold Standard for quantitation because of MRMIf Q1 d Q3 idth 0 th MRMIf Q1 and Q3 width=0, then MRMMany (hundreds!) of precursor to product ion pairs can be

it d (A B A’ B’ A” B” t )monitored (A-B, A’-B’, A”-B”, etc.)MRM analysis is the best way to maximize signal intensity of product ions

MS/MS - Multiple Reaction Monitoring (MRM)

Precursor ion set

Product ion set

Fragmentation(CAD)set set(CAD)

MS/MS Systems: Quadrupole-TOF (QTOF)

Introduced commercially by Micromass around 1995.Brilliant innovation first commercial hybrid MS/MSBrilliant innovation, first commercial hybrid MS/MS.They charged “what the market would bear”

$500-600kNo competition!Great qualitative analyzer

TOF analyzer provided:TOF analyzer provided:Incredibly fast scan ratesAccurate mass capability (MW confirmation)Higher resolution (8k initially, now 10-12k w/ 1 reflectron)

QSTAR® XL System—Schematics

DC Quad250 L/s770 L/s

Acceleratorcolumn

4-anode detector

column

Sample

Ions

Conductingliner770 L/s

2.5 Torr

Q1

10-2 Torr

10 mTorr

C t iLINAC

Q2Q0

10-2 Torr

Field Free

Drift region

7x10-7 TorrEffective Flight

Curtain

Gas

• eliminate cross-talk

Ion Mirror

(reflector)

Path = 2.5 m• fast switching MS MS2

• broad dynamic range

• saturation correction

Micromass QTOF Premier

What can a QTOF do for you…?

4 “Types” of Ion Traps…

Analyzer Types: Ion Traps -MS/MSn Systems

3D Ion Trap

3D Ion Trap- MS/MS Operation

Triple Quads vs. Ion Traps Complementary MS/MS Approaches:

Tandem in Space: Triple QuadsTandem in Space: Triple QuadsPoor scanning sensitivity

Great for quant (MRM)Great for quant (MRM)

Very selective scans

T d i Ti I TTandem-in-Time: Ion TrapsVery sensitive scanning

O l d t iOnly product ion scans

Only scanning capability

I T 2D (2002)Ion Traps -2D (2002)“Ion bottles” for optical spectroscopyspectroscopy.

Minimize fringing fields to maximize performance.

Ion accumulation forIon accumulation for enhanced ms sensitivity.High quality mass

t tspectrometer:RCM, 2002, 16, 512-526.

3 D TLinear Traps

Feb. 1, 20073-D Traps

AB-Sciex Q TRAP™ System Ion Path

N CAD GDipolar Aux AC

N2 CAD GasSkimmer

Q0 Q1 Q2 Q3

ExitIQ3IQ2IQ1O ifi

linear ion trap

3 4x10-5 Torr

ExitIQ3IQ2

LINAC

IQ1Orifice

3-4x10-5 Torr

Trapping Forces in a Linear Ion Trap

Radial Trapping RF Voltage

AxialTrapping

AxialTrapping pp g

DCVoltage

Exit Lens

Radial Trapping RF VoltageResonance Excitation

Linear vs. 3-D Ion Traps:Li TLinear Trap

Trapping EfficiencyNo quadrupole field on center

3-D TrapTrapping Efficiency

Q d l fi ld i lit d No quadrupole field on center line.Longer flight path.

Quadrupole field gives amplitude and phase dependent injection eff’s.

1 cm to lose injection energy~1 cm to lose injection energy.

Extraction EfficiencyLinear trap is ~10X better

Extraction Efficiency18-20% (measured)

< 50% (Probably ~30%)

I C it3 D trap is 2X betterIon Capacity:

Ion Capacity:3-D trap is ~2X better

5-inch linear trap:5 inch linear trap:

45X greater capacity

MSn Systems: FTMS Ion Trap- Orbitrap

Finnigan LTQ Orbitrap FTMS

Feb. 1, 2007

Finnigan LTQ Orbitrap FTMS

Orbitrap (Brochure) Data

MSn Systems: FTMS (ICR) w/2D Ion Trap Front End…

Thermo FTMS (ICR)…

Feb. 1, 2007

FTMS Data Example…

ABI-4800 TOF-TOF

3D Ion Trap - TOF New Hybrid Design

Shimadzu “LCMS IT-TOF”

Summary

Role and importance of MS in bioanalytical analysis continues to grow and evolve

MS is typically no longer the bottleneck (sample handling and data acquisition/processing slow thru-put)Power of new MS technology providing new dimension of information on biomoleculesgy p g

There are 3 fundamental MS analyzer technologies, each with it’s advantages and disadvantages.

‘H b id ’ t k d t f b t f 2 t h l i‘Hybrids’ can take advantage of best of 2 technologiesLC/MS continues to evolve at a rapid rate

Better, faster, cheaper…, , p