Agilent Restricted1

CE Capillary

N2Gas

Sheath Liquid

Microsampling/DBSLatest Innovations from Agilent Technologies

CE-MS Chip-MS DBS-MS

Pre Extraction Image

April 13, 2023

New Polaris-Chip for highest resolution

Stationary Phase

Enrichment column

Sprayer-tip

Analytical column

Inert polyimide

• Polaris C18-A, polar embedded phase,

3 µm, 180 Å, 200 m2/g

• 150 mm x 75 µm ID

• 1 µg sample loading

Loading capacity for new Polaris Chip

• Shown: E.coli TIC and EIC‘s of 500 ng injection superimposed

• Determined using 9 representative peptides across elution range

2x10

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+ESI EIC(858.7801) Scan Frag=175.0V Load_500.d

Counts (%) vs. Acquisition Time (min)6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

P1 P2 P3 P4 P5 P6 P7 P8 P9

April 13, 2023

Agilent CE-ESI-MS Setup

MassHunter SW control:

- CE instrument- MS instrument- LC makeup flows

Page 5

Small anionic molecules in Metabolomics by CE-ToF-MS

CE-MS is ideally suited to handle ionic or polar metabolites in complex mixtures reproducibly

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Dried Blood Spots (DBS) Analysis

• µLs sample• Stable on card at ambient temp• Easy to ship• Non-biohazardous

• mLs sample• Must store frozen• Difficult to ship• Biohazardous

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An Automated Online Card Extraction

LC/MS System for Dried Blood Spots

Analysis

DBS CardRacks

Robotic Gripper

SwitchingValvesClamp Module

Camera

ISTD Dilutor

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Agilent Automated Card Extraction (AACE) LC/MS System Flow Schematic

Clamped DBS Card

10-PortValve

6-PortValve

Extraction Pump (1)1260 Binary LC

Dilution Pump1200 Isocratic LC

Analytical Pump (2)1260 Binary LC

ISTD

ISTDLoop

Trap 1 Column

Trap 2 Column

Analytical Column

A B A A BWaste

6460 QQQMaster Control

Prolab Card Extration Instrument

Agilent Restricted9

MRM chromatograms of clozapine, noreclozapine, and clozapine-N-oxide

Reproducible and sensitive DBS/MS systemLOQs for all compound 0.5 ng/mL in blood

Clozapine Norclozapine Clozapine-N-oxide

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Summary: AACE System vs. Offline DBSComparable Quantitation Performance Parameters

AACE LC/MS System

Offline Extraction DBS

CompoundLOQ

(ng/mL in blood)Range

(ng/mL)Linearity

(R2)Accuracy

(%)Reproducibility

(%)Precision

(%)

Clozapine 0.5 0.5 – 10000 0.9996 92.1 – 106.8 0.61 – 5.61 4.82

Norclozapine 0.5 0.5 – 5000 0.9988 85.2 – 109.9 0.84 – 6.05 8.94

Clozapine N-oxide 0.5 0.5 - 5000 0.9981 93.2 – 104.1 0.68 – 6.15 3.82

Analysis Time ~ 1 hr

CompoundLOQ

(ng/mL in blood)Range

(ng/mL)Linearity

(R2)Accuracy

(%)Reproducibility

(%)Precision

(%)

Clozapine 0.5 0.5 – 10000 0.9997 88.0 – 103.2 0.61 – 8.92 4.89

Norclozapine 0.5 0.5 – 10000 0.9991 89.3 – 107.1 0.08 – 8.98 8.36

Clozapine N-oxide 0.5 0.5 - 10000 0.9991 91.8 – 108.4 0.25 – 8.66 6.64

Analysis Time ~ 5 hrs

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DBS Summary – fast and “fit for purpose”

Agilent Automated Card Extraction (AACE) • Automated flow-through DBS analysis • Fully integrated with MassHunter LC/MS

Data Processing and Reporting• Pre- and post- sampling DBS card images• Processed quantitative data• Chain of custody

Clozapine study • Sensitivity: 0.5 ng/mL LLOQ • Linear assay over 4 orders of magnitude• Comparable results with offline method• Time savingss, 1 hour versus 5 hours