Advanced Chemistry Development, Inc. (ACD/Labs)

n-Dimensional MAP Chromatography:

Virtual Resolution of Components Based on

Orthogonal Chromatographic Methods

M. McBrien, A. Galin, A. Vazhentsev, and Vitaly Lashin

Pittcon – February 25/07

2

Outline

Why match peaks?Reviewing MS-MAPOrthogonal separations and chromatographic screeningPulling it all together – NDMC and “virtual resolution”Limitations of the MAP algorithmsThe future

3

Typical Chromatographic MethDev

Optimization of continuous variables –i.e., concentration of organic modifierSelectivity changes are modest; typically you are “tweaking” a promising method

4

Automated Method Development? The Challengetfa series.esp

605550454035302520151050Retention Time (min)

12

34

56

78

910

1112

1314

1516

Go from a series of collected datafiles under different conditions…

5

Automated Method Development? Peak Matching

90858075706560555045403530

Solvent B, %

6

8

10

12

14

16

18

TFA

, mM

3.893.643.403.162.912.672.432.191.941.701.461.210.970.730.490.240.00

…to a model of the sample’s behavior over any value of those conditions:

6

Peak Matching

Perhaps the greatest challenge in computer-assisted method development.All peaks must be tracked across all experimentsHumans use spectra, intuition, and test injections.Computers are more limited.

7

Matching Peaks for Complex Work

Each component in a separation must be tracked

Manual peak tracking can be tedious in CA MethDev; it’s impossible in

AMD!min6 8 10 12 14 16 18 20

mAU

0

25

50

75

100

125

150

175

min6 8 10 12 14 16 18 20

mAU

0

25

50

75

100

125

150

175

YMC Pro C18 pH 2.5

XTerra RP18 C18 pH 7

8

MS-Mutual Automated Peak Matching

MS offers significant advantages over UV:Greater resolutionS/NOrthogonal signal strength

The MS-MAP algorithm was designed to take advantage of the differences in these detectors

9

Preparing for Peak Matching -IntelliXtract

10

IntelliXtract Key Features

Built on CODA peak extraction technology, but takes it to the next level Uses a mixture of

Chemometrics (CODA, Mass Cluster Analysis, Isotopic Matching…), andMass Spectral knowledge

Follows the same logic that an expert would use for manual data interpretationA self-optimizing algorithm

Software parameters are calculated dynamically

11

DefinitionsComponent

A component is defined as a single chemical entity eluting in chromatographic space

5.04.54.03.53.02.52.01.51.00.5Retention Time (min)

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95

1.00

Rel

ativ

e In

tens

ity

TIC

Extracted component

[M+H]+

12

IntelliXtract

Examine each mass-peak individuallyValidate chromatographic character of peakDesignate each peak’s “role”:

(M+H)+

AdductDimer/trimerProbable fragment ion (PFI)Isotopes

13

MS-MAP: Reconciling Components

Perfect co-elution is a problem for IX (in a single experiment)Sometimes, M+H+ assignment can be wrongSolution:

Full spectral matching post-IXLogic-based reconciliation (if new component arises in run2, then re-examine run1’s PFIs)Orthogonal detection (UV-MAP)

14

Reconciliation Logic

1. From IX:Masses for each tR and each expt. with (M+H)+

label2. Match all “complete” (M+H)+s spectra3. Search all missing (M+H)+ masses for PFI or

adduct assigned peaks in other peaks4. Transfer peak information where masses are

found5. Label redundant masses6. Reconcile with LC/UV data

15

Illustrating MS-MAP

16

NONAME00

Using a second experiment to supplement IX

Comp. A

Comp. B

288289

287

365365366366

320321

Component A and B coelute perfectly in expt. 1. IX will assign the component A masses “probable fragments”.

17

NONAME01

NONAME00

Using a second experiment to supplement IX

Comp. A

Comp. B

288289

287

365365366366

320321

The emergence of the new pattern enables us to track the component from expt2 back to expt 1.

18

NONAME19 1

NONAME17

NDMC “Virtual Resolution”

Comp. A

Comp. C

Comp. B

288289

287

365365366366

320321

303

305305304

• Component A has chromatographic resolution in experiment 1.• Component B has CR in exp 2.• Component C is resolved only through the use of both expts!

19

Virtual Resolution

A component needs be resolved from its partner in only one experiment to be tracked, and need never be resolved from all other components in even one single run.

20

GC x GC of yeast cell extract

21

NDMC Projections – Orthogonal Methods with Peak Tracking

Collect orthogonal dataMatch peaksEstablish peak table:

Component, tR, Width, etc.Widths and tRs in any two experiments can be combined

22

2 Dimensional MAP Chromatography

NONAME19 1

Comp. A

Comp. C

Comp. B

288289

287

365365366366

320321

303

305305304

NONAME17

NONAME17

23

2D MAP Chromatography

Coelution in method 2

Coelution in method 1

24

NONAME19 1

NONAME17

NONAME17

NONAME17

NONAME17

NONAME17

NONAME17

…more experiments

NONAME17

Comp. A

Comp. C

Comp. B

288289

287

365365366366

320321

303

305305304

25

NONAME19 1

NONAME17

NONAME17

NONAME17

NONAME17

NONAME17

NONAME17

…more experiments

NONAME17

Only in the 8th

experiment were comps A and C

resolved.

26

NONAME19 1

NONAME17

NONAME17

NONAME17

NONAME17

NONAME17

NONAME17

…more experiments

NONAME17

In this case, 8 experiments were required in order

to resolve component C!

27

Resolving Power of Multidimensional Chromatography

28

Adding Dimensions

Any peak-matched experiments can be used to contribute to the component tableResolution of a given pair of components is necessary in only one experimentThe more (orthogonal) experiments, the greater the chance of virtual resolution

29

Theoretical Peaks

~100 peaks in 1D HPLC?2D MAP HPLC: 10000 peaks32D: 1064 peaks

)ln(4

1M

RC t

tNn +=

Three very important assumptions are made:• Signals are independent of coelution• Retention times don’t correlate• No isobaric compounds…therefore, in practice, far fewer peaks will be resolved.

30

How best to design the chromatographic conditions?

Ideally, the chemical environment is stable, but the chromatographic environment changes dramatically.Logically, the column is the thing to change.

31

Chromatographic Conditions and Peak Matching

MAP works best with smallsolvent condition changes:

ColumnColumnSolvent strengthSolvent strengthTemperatureTemperatureSolventSolventpHpH

32

Some Early NDMC ResultsMethod Development Suite LC/MS 10.0

2-Dimensional MAP Chromatography:Acetic acid gradient methanol vs. acetonitrileGeneric gradients; no optimization of chromatography or detector

10 compound set:m-toluamideUmbelliferonePropazineMirtazapineDexamethasone4-aminobenzoic acidBenzoylecognineLinomycinPrilosec4-hydroxy-3-methoxyphenylacetic acid

33

Some Early NDMC Results (1)

2-Dimensional MAP Chromatography:Waters Alliance 2690 with ZQ Single Quad MS Acetic acid gradient methanol vs. acetonitrileGeneric gradients; no optimization of chromatography or detector

10 compound set:m-toluamideUmbelliferonePropazineMirtazapineDexamethasone4-aminobenzoic acidBenzoylecognineLinomycinPrilosec4-hydroxy-3-methoxyphenylacetic acid

Failed to ionize

Correctly identified (M+H)+

34

Some Early NDMC Results (2)

6-Dimensional MAP Chromatography:

Waters Alliance 2690 with ZQ Single Quad MS Supelco C18/Amide/Phenyl Columns:

5 cm x 4.6 mm ID 5 micron0.1% Acetic acid, ammonium acetate, 5 min acetonitrile gradient 5%-95%Generic gradients; no optimization of chromatography

Sample set:59 non-isobaric compounds: 10 ug/mL Commercially available107.07 < MW < 853.33All known to give signal when resolved under these detector conditions

35

Standards Set Results2DMC (average)

Of 59 compounds:33 found with correct MW, tRs23 false positives26 unmatched

36

Standards Set Results3DMC (average)

Column was the variable changedBuffer was held constantOf 59 compounds:

37 found with correct MW, tRs6 false positives22 unmatched

37

Standards Set Results6DMC

Column and mobile phase changeOf 59 compounds:

42 found with correct MW, tRs5 false positives17 unmatched

Note:Total Peak width = 13.7 minutesEffective Chr’gram ~ 5.5 minutesOn average, almost 3 components coeluteat any point in the ch’gram.

38 tR amide/acetic acid

tRC18/ acetic

acid

MAP Projection6-DMC

39

MAP Projection6-DMC

tR C18/ammonium acetate

tRAmide/

acetic acid

40

The value of added dimensions

A few extra components were locatedMany false positives were excluded

41

Tannins Project

Ontario spruce tannins:High MW fraction was collected

Instrument:Agilent 1100, series pump autosampler, pump (quat), degasser.Detectors:

Bruker DADHP/Bruker Esquire LC 3D ion trap MS (negative mode)Additional acetonitrile ~20uL/min was added by a Bruker NMR-MS interface (BNMI) just prior to MS to aid electrospray.

42

Chromatographic Conditions

Gradient:1) Isocratic 90:10 (Water:acn) for 5 mins2) Linear Gradient 5-60mins (Water : acn) to 10:903) Linear Gradient 60-80mins (Water : acn) to 0:1004) Isocratic 0:100 (Water:acn) from 80-100mins

4-dimensional MAP Chromatography:Xterra PhenylAscentis AmideSupelco PFPPAscentis C18

43

Ascentis RP-Amide

Supelco-PFPP

888072645648403224168Retention Time (min)

Ascentis C18

Xterra Phenyl

The TICs

44

96 unique mass components found38 isobaric sets

NDMC Projection

tRC18

tR Amide

45

How Many Dimensions?

Large numbers of sets of somewhat orthogonal HPLC conditions can be designedThe resolving power of NDMC is limited by the number of sets of orthogonal conditions that still allow for consistent spectrapH and column are primary dramatic selectivity modifiersLots of stationary phases exist, but eventually, they become redundantSpectral matching with pH changes will likely be challenging to perfectIsobaric compounds will still be a challenge in LC

46

Where can NDMC be useful?

COMPLEX SAMPLESInitial investigation of (unknown composition) samplesAs a fast detection technique for preliminary detection of componentsAs part of initial screening of the sample for detection of impuritiesIn supplement to manual LCMS analysis

47

Some Remaining Questions

Where is the best compromise between run time and added dimensions?

Suppression of ionizationEfficiency of peak matching

How can we further reduce false positives?How to reduce missed components?

composite samples…it is possible to combine multiple detector settings automatically…

48

Conclusion

Orthogonal chromatographic techniques combined with MAP:

Method design toolAutomated detection device

A useful tool for complex sample investigation with no prior knowledge of componentsRequires no specialized hardwareRidiculous theoretical resolving power is practically limited

49

Acknowledgements

ACD/Labs:A. Bogomolov and A. Galin

Supelco:David BellJacinth McKenzie

University of Toronto:Andre Simpson

University of WashingtonRob Synovec