ASB-C18 Performance and Applications

• Surface Deactivation and Silanol Enrichment：

SiO2---OH

Unisol Technology

Si(OMe)4

SiO2---O-Si(OMe)3

Hydrolysis

SiO2---O-Si(OH)3

R-Si(Me)2-XSiO2-O-SiOSi(Me)2R

Results from the Unisol Process• Metal on silica surface----covered by fr

esh silanol of low acidity• High acidity of silanols caused by high

temperature sintering---- covered by fresh silanol of low acidity

• Surface irregularity caused by formation of micro-crystalline domains on surface ---- more uniform surface

Products from Unisol Technologies

• Unisol C18: true versatile RP columns to cover the broadest range of applications; the most friendly towards basic or acid compounds; high Aq compatible

• Venusil ASB C18: ASB-C8: extremely low bleed and high sensitivity for MS; extremely low pH and high temperature tolerance (pH=0.8, Temp=100oC); high Aq compatible

• Unisol Amide: the highest retention for hydrophilic compounds in HILIC mode; more stable and reproducible than silica or amino columns

• Durashell RP: the broadest pH range (1-12); symmetric peaks for acidic, basic and neutral compounds

Venusil ASB-C18• Very polar C18• pH range 0.8-7.0 (extremely stable at pH=1, up to

100oC) • Non-endcapped C18, • 150 A, 200 m2/g (compared to Zorbax 80A, 180

m2/g)• Silanol pH=5.2 (compared to Zorbax pH=3.5)• Compatible to 100% water (compared to Zoba

x SB, non-compatible)• Highest LC-MS Sensitivity : 2-3 times higher t

han many popular brands (low bleed, inert surface and high efficiency)

Peak Shape of Basic Compounds

Test conditions:Column dimension: 4.6 mm x 150mmSample: doxepin, nortriptyline, trimipramine amitriptylineMobile phase: Acetonitrile/0.01M sodium phosphate (pH=5) = 70/30Temperature: 30 oCFlow: 1 mL/minDetection: UV 254 nm

Brand A C18( non-endcapped, )

C18 (non-endcapped, twin-layer

Sensitivity Comparison for LC-MS2.5 ng/mL Pseudoephedrine

Agela Venusil ASB C18, 2.1×50mm, 5µm

Aglent Zorbax XDB C18, 2.1×50mm, 5µm

Phenomenex Luna C18, 2.0×50mm, 5µm

Best S/N:X2-3 sensitivity comparing to other columns

Agela Venusil ASB C18, 2.1×50mm, 5µm

Aglent Zorbax XDB C18, 2.1×50mm, 5µm

Phenomenex Luna C18, 2.0×50mm, 5µm

Results Obtained Under Same LC-MS Conditions0.01 ng/mL Pseudoephedrine

Best S/N:X2-3 sensitivity comparing to other columns

Quantitative Analysis of oleic acid and Its MetQuantitative Analysis of oleic acid and Its Metabolite Using SPE and LC-MS abolite Using SPE and LC-MS

Existing Issue

• Protein precipitation has low recovery: ~ 10%• Poor linearity in the experimental range

I.S.

Oleic Acid

metabolite

Venusil ASB C18 4.6mm×150mm

Oleic acid

I.S.

Sample Preparation Two: 1 mL PEP SPE Columns

Load500uL Sample

Wash 11mL 1% formic acid

Elute1 mL 1% formic acid in ACN

LC-MS

Equilibration 1mL Water

Condition 1 mL Methanol

Concentration10ng/mL(n=3)

100ng/mL(n=3)

2500ng/mL(n=3)

Oleic acid 77 87 91

RSD 2.9 0.9 0.2

Added 3% Phosphoric acid to break the protein-drug binding

Still non-ideal recovery for Oleic Acid

Optimization of the Elution Solvents

Test concentration: 100ng/mL n = 12

80:20 70:30 60:40 50:50

Oleic acid Recovery

93 98 91 81

RSD 3.8 2.9 4.6 3.5

DHSARecovery

85 95 83 76

RSD 4.9 3.5 4.2 4.1

The ACN:MeOH=70:30 solvent mixture gives the best recoveries for both oleic acid and its metabolite DHSA. Strong Interaction of the molecules with the SPE sorbent requires stronger elution solvent.

Recovery of compounds in Plasma

Establishing a High Efficient, High RecoEstablishing a High Efficient, High Recovery SPE Method and a Fast, Sensitive very SPE Method and a Fast, Sensitive

HPLC-MS/MS Method HPLC-MS/MS Method

SPE and SPE and 2.1 x 50 mm HPLC Columns2.1 x 50 mm HPLC Columns

0.7mL/min利多卡因

0.7mL/min伪麻黄碱

Lidocaine

Pseudoephedrine

Establish a Fast LC-MS/MS Quantitation MethodUsing Agela Venusil ASB C18 Column, 2.1mm×50mm, 5 um

Internal Standard

• Instrument:– API Qtrap 3200, Applied Biosyste

m– LC-20AHPLC, Shimazu

• MS Conditions: – ESI, Positive ion mode, MRM

m/z 166.0 m/z 148.1(Pseudoephedrine)

m/z 235.3 m/z 86.1(internal standard: Lidocaine)

• HPCL ConditionsColumn: 2.1 x 50 mm, 5 um, ASB C18A: 0.1% Formic acidB: MethanolFlow rate: 0.5 mL/minGradient:20%-95% B in 2 min5%,hold at 95%B for 2.5min,decrease to 20% B at 3 min, and hold for 0.5 min. Total analysis time 3.5 minutes.

Load500uL Sample

Wash 11mL 1% formic acid

Elute1 mL 1% formic acid in ACN

LC-MS

Equilibration 1mL Water

Condition 1 mL Methanol

Condition 1 mL Methanol

1mg PEP

Add 3% Phosphoric Acid to Break Compound/Plasma protein binding

Consider to Use PEP SPE Columns

Std curve of wei ma huang jian in plasma

Recovery of the Plasma Samples Using 1 mL PEP SPE Columns

10ng/mL(n=3)

100ng/mL(n=3)

2500ng/mL(n=3)

PseudoephedrineRecovery

98 96 100

RSD 2.6 0.7 0.2

Lidocaine Recovery

89 93 97

RSD 2.9 1.7 0.3

Ideal recoveries are obtained using PEP SPE columns