2x4 Method Development


2x4 Method For µElution Plates

	Sample volume range	10 - 375 µL
	1) What sample volume will you use for this Method (µL)		µL
	2) What is the mid-point of the concentration range for this method?		ng/mL



	Method Details
			MCX & WAX	MAX & WCX
	Plasma (sample) volume
	Pre-SPE Treatment (diluent) volume

	Condition
	Equilibrate
	Load
	Wash
	Elute 1
	Elute 2
	Total Elution volume per Elute
	SPE Concentration or Dilution Factor
	Flow Rate for SPE method

	Oasis format chosen	96 well Plate
	Format details	2mg µElution Plate
	Oasis Sorbent Selected	Method Development Plate
	Waters Part Number to Order			© Copyright Waters Corporation 2012

	The µElution format has been selected to avoid the evaporation step for compounds such as Peptides, small Proteins, volatile Compounds and/or heat labile Compounds.
	It also allows sample concentration up to x15 without evaporation


2x4 IEX µElution Plates Recovery and Matrix Effects Experimental Set-up
	Mid-point of the concentration range for this method?		ng/mL
	Number of Data points (replicates)	3	3-pre SPE & 3-post SPE spike
STANDARDS PREPARATION
	Concentrated Standard Stock Solution preparation (S1)


	Diluted Standard Solution preparation (S2)





PLASMA PREPARATION
	Approximate amount of bulk Plasma needed for these experiments
	Pre-Spike Plasma preparation (P1)







	Final Concentration of (P1)		ng/mL
	Post-Spike Plasma preparation (P2)






				© Copyright Waters Corporation 2012
Pre-Treatment (dilution) Preparation
	Base			Acid

2X4 Experimental Set-up

MeOH

Condition

µL

Water

Equilibrate

µL

P1 + 4% H3PO4 (Acid)

P1 + 4% NH4OH (Base)

Load

µL

P2 + 4% H3PO4 (Acid)

P2 + 4% NH4OH (Base)

Load

µL

2% Formic Acid in Water

5% NH4OH

Wash

µL

1

2

3

4

5

6

7

8

9

10

11

12

1

2

3

4

5

6

7

8

9

10

11

12

Methanol

Elute 1

µL

A

5% NH4OH in 60/40 ACN/MeOH

2% Formic Acid in 60/40 ACN/MeOH

Elute 2

µL

B

C

↓

D

Elute 1

→

E

→

E

1

2

3

4

5

6

7

8

9

10

11

12

F

A

G

B

H

C

COLLECT LOAD

Collection Plate for Elute 1

D

AND WASH Steps

Add Water

50 µL

Add Standard (S1)

50 µL

E

( see 2x4

Add standard (S2)

50 µL

Then add Methanol

50 µL

F

troubleshooting )

1

2

3

4

5

6

7

8

9

10

11

12

1

2

3

4

5

6

7

8

9

10

11

12

G

P1+A

P1+B

A

H

P2+A

P2+B

B

MCX

WAX

MAX

WCX

C

D

→

D

Elute 2

→

E

F

G

H

Collection Plate for Elute 2

Add Water

50 µL

Add Standard (S1)

Add standard (S2)

50 µL

Then add 5% NH4OH in

Then add 2% Formic Acid in

60/40 ACN/MeOH


2x4 Results for Recovery and Matrix Effects
	1	2	3		4	5	6		7	8	9		10	11	12
A																	Results for Elute 1
B																	Reversed Phase bound components
C																		MCX		WAX		MAX		WCX
D																	Recovery		%		%		%		%
E				Avg				Avg				Avg				Avg	Matrix Effects		%		%		%		%
F																	Matrix Factor
G
H
	MCX				WAX				MAX				WCX				ENTER LC/MS results into their exact location in the collection plates
	Collection Plate for Elute 1																All 3 values need to be entered

	1	2	3		4	5	6		7	8	9		10	11	12
A																	Results for Elute 2
B																	Ionic bound components
C																		MCX		WAX		MAX		WCX
D																	Recovery		%		%		%		%
E				Avg				Avg				Avg				Avg	Matrix Effects		%		%		%		%
F																	Matrix Factor
G																				© Copyright Waters Corporation 2012
H
	MCX				WAX				MAX				WCX
	Collection Plate for Elute 2

Understanding Charge States during

OASIS Ion Exchange Solid Phase Extraction

Method Step

Load

Wash

Elute 1

Elute 2

INPUT HERE

Compound Type

compound

sorbent

compound

sorbent

compound

sorbent

compound

sorbent

pH when loading

ACIDS (pKa < 1)

Sorbent

ACIDS (pKa 2-8)

Protocol

BASES (pKa >10)

BASES (pKa 2-10)

NEUTRALS

ZWITTERIONS

Key

Wash

Elute 1

Elute 2

Negative Charge

-

Neutral Charge

NC

Eluted Compounds

Postive Charge

+

1

2

3

4

5

6

7

8

9

10

11

12

1

2

3

4

5

6

7

8

9

10

11

12

A

B

C

D

LOAD

D

E

STEP

E

F

→

F

G

P1+A

P1+B

G

H

P2+A

P2+B

H

MCX

WAX

MAX

WCX

Collection Plate for LOAD AND WASH STEPS

Rotate Plate

҉

↓

WASH STEP

IT IS IMPORTANT TO COLLECT THE LOAD

AND WASH STEPS FOR TROUBLESHOOTING

H

FOR POOR RECOVERY RESULTS

G

F

E

D

C

B

A

12

11

10

9

8

7

6

5

4

3

2

1

Collection Plate for LOAD AND WASH STEPS


Analyte is found in:	Possible cause	Suggestion to change method	Example of change
Load step (breakthrough)	Analyte still bound to protein	Change dilution solvent / modifier	try 1:1 dilution with aqueous 4% H3PO4 solution
		Change dilution solvent / modifier	try 1:1 dilution with aqueous 5% NH4OH solution
	Matrix overload	Reduce amount of sample loaded	eg change from 500 ul to 200 ul plasma loaded
		Increase amount of sorbent used	eg change from 10 mg to 30 mg
	Analyte is too polar for the pH in load step	Change pH of sample diluent in Wash 1 protocol	Swap aqueous 5% NH4OH with aqueous 4% H3PO4 or vice versa
Wash 1 (aqueous)	Analyte is too polar for the pH in load step	Change pH of sample diluent in Wash 1 protocol	Swap aqueous 5% NH4OH with aqueous 4% H3PO4 or vice versa
Wash 2 (organic)	Non-polar analyte unretained at pH in Wash 1	Change pH of Wash 1 according to protocol
Analyte not found	Analyte is still on the sorbent	Use stronger elution solvent	eg Acetonitrile instead of MeOH
		Use more elution solvent	eg 2 x 50 ul instead of 2 x 25 ul
	Analyte is not soluble in elution solvent	change elution solvent according to literature solubility
	Analyte is labile and has been 'breaking down'	Check literature for stability and adjust	eg change from MCX to WCX with base labile compounds

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