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ASB-C18 Performance and Applications

ASB-C18 Performance and Applications. Unisol Technology. Surface Deactivation and Silanol Enrichment ： SiO2---OH. Si(OMe) 4. SiO2---O-Si(OMe) 3. Hydrolysis. R-Si(Me) 2 -X. SiO2-O-SiOSi(Me) 2 R. SiO2---O-Si(OH) 3. Results from the Unisol Process.

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ASB-C18 Performance and Applications

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  1. ASB-C18 Performance and Applications

  2. Unisol Technology • Surface Deactivation and Silanol Enrichment： SiO2---OH Si(OMe)4 SiO2---O-Si(OMe)3 Hydrolysis R-Si(Me)2-X SiO2-O-SiOSi(Me)2R SiO2---O-Si(OH)3

  3. Results from the Unisol Process • Metal on silica surface----covered by fresh 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

  4. 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

  5. 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, 180m2/g) • Silanol pH=5.2(compared to Zorbax pH=3.5) • Compatible to 100% water(compared to Zobax SB, non-compatible) • Highest LC-MS Sensitivity : 2-3 times higher than many popular brands (low bleed, inert surface and high efficiency)

  6. Peak Shape of Basic Compounds C18 (non-endcapped, twin-layer Brand A C18( non-endcapped, ) Test conditions: Column dimension: 4.6 mm x 150mm Sample: doxepin, nortriptyline, trimipramine amitriptyline Mobile phase: Acetonitrile/0.01M sodium phosphate (pH=5) = 70/30 Temperature: 30 oC Flow: 1 mL/min Detection: UV 254 nm

  7. Sensitivity Comparison for LC-MS 2.5 ng/mL Pseudoephedrine 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

  8. Results Obtained Under Same LC-MS Conditions 0.01 ng/mL Pseudoephedrine 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

  9. Quantitative Analysis of oleic acid and Its Metabolite Using SPE and LC-MS

  10. Existing Issue Protein precipitation has low recovery: ~ 10% Poor linearity in the experimental range Oleic acid I.S. I.S. metabolite Oleic Acid Venusil ASB C18 4.6mm×150mm

  11. Sample Preparation Two: 1 mL PEP SPE Columns Condition 1 mL Methanol Equilibration 1mL Water Load 500uL Sample Wash 1 1mL 1% formic acid Elute 1 mL 1% formic acid in ACN LC-MS Added 3% Phosphoric acid to break the protein-drug binding Still non-ideal recovery for Oleic Acid

  12. Optimization of the Elution Solvents Recovery of compounds in Plasma 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. Test concentration: 100ng/mL n = 12

  13. Establishing a High Efficient, High Recovery SPE Method and a Fast, Sensitive HPLC-MS/MS Method SPE and 2.1 x 50 mm HPLC Columns

  14. Establish a Fast LC-MS/MS Quantitation Method Using Agela Venusil ASB C18 Column, 2.1mm×50mm, 5 um • Instrument: • API Qtrap 3200, Applied Biosystem • 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) Pseudoephedrine 0.7mL/min 伪麻黄碱 • HPCL Conditions • Column: 2.1 x 50 mm, 5 um, ASB C18 • A: 0.1% Formic acid • B: Methanol • Flow rate: 0.5 mL/min • Gradient: 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. Lidocaine Internal Standard 0.7mL/min 利多卡因

  15. Consider to Use PEP SPE Columns 1mg PEP Condition 1 mL Methanol Add 3% Phosphoric Acid to Break Compound/Plasma protein binding Equilibration 1mL Water Load 500uL Sample Wash 1 1mL 1% formic acid Elute 1 mL 1% formic acid in ACN LC-MS Std curve of wei ma huang jian in plasma

  16. Recovery of the Plasma Samples Using 1 mL PEP SPE Columns Ideal recoveries are obtained using PEP SPE columns

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