Standards and IS, Clarity OTX Central Nervous System (CNS) Tissue

LC Conditions

Column

bioZen 2.6 µm Oligo, LC Column 100 x 2.1 mm, Ea

Brand

Biozen LC

Part No

00D-4790-AN

Phase Name

Oligo

Elution Type

Gradient

Mobile Phase

A: 1.0 % HFIP, 0.1% DIPEA in Water with 10 µm EDTA

B: 0.075% HFIP, 0.0375% DIPEA in Water:ACN (35:65) with 10 µm EDTA

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

95

5

2

0.1

95

5

3

2.5

80

20

4

4

40

60

5

4.1

5

95

6

4.4

5

95

7

5

80

20

8

5.8

5

95

9

6.1

5

95

10

6.2

80

20

11

6.3

20

80

12

7.5

5

95

13

7.8

5

95

14

7.9

95

5

Flow Rate

0.50 mL/min

Temperature

80°C

System

Shimadzu Nexera UHPLC SCIEX 6500+

Detection

LC/MS/MS

Detection Info

Sample Preparation Details

Description

Clarity OTX 100 mg Plate, 100 mg/well 96-well plate, Ea

Part No

8E-S103-EGA

Pretreatment Note

Aliquot 75 µL calibration standards, QC samples, study samples onto 2 mL 96-well round bottom plate Add 30 µL IS (1 µg/mL) to all wells except blank controls (add 30 µL water to blanks) Condition 96-well plate with 1 mL MeOH Equilibrate with 1 mL Equilibration Buffer (50 mM Ammonium Acetate, pH 5.5) Add 600 µL of Lysis Loading Buffer to all samples Vortex mix samples for ~5 min; incubate at RT for additional 5 minutes Transfer solution to TOMTEC or equivalent Wash OTX plate 3X with 1 mL Equilibration Buffer Wash plate 3X Wash Buffer (50 mM Ammonium Acetate, pH 5.5: ACN, 50:50) Add 0.5 mL Elution Buffer (100 mM Ammonium Bicarbonate, pH 9.5THF (50:40:10, v/v/v) and elute into collection plate Dry samples to ~400 µL by Nitrogen

Condition 1

Methanol

Load

Pre-treated sample

Wash 1

50 mM Ammonium Acetate, pH 5.5/50% ACN

Elution 1

100mM Ammonium Bicarbonate, 1mM TCEP, pH 9.5/40 % THF

Evaporation

Evaporate to dryness

Steps

0

Condition

Methanol

1

Equilibrate

50 mM Ammonium Acetate, pH 5.5

2

Load

Pre-treated sample

3

Wash

50 mM Ammonium Acetate, pH 5.5/50% ACN

4

Elute

100mM Ammonium Bicarbonate, 1mM TCEP, pH 9.5/40 % THF