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digoxin_lc.md

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Analytical procedure for automated LC/MS/MS assay of digoxin and dihydrodigoxin in plasma

Modified from Yong Huang 6/16

Version 1.0 16 May 2018 ENB + JB

Materials

Reagents, consumables, and equipment

  • HPLC Grade Methanol
  • MQ water
  • Ethyl Acetate, 0.5 mL per sample
  • Methyl t-butyl ether, 0.5mL per sample
  • Acetonitrile
  • Glass pasteur pipettes (~1mL), 1 per sample
  • 16x48mm test tubes (Agilent 5022-6533), 2 per sample
  • Autosampler vials (485050 E&K scientific)
  • Turbovap and nitrogen tank
  • Appropriate serum blank (germ-free mouse serum)
  • Dry ice and methanol/isopropanol bath
  • Vortex
  • Fume Hood
  • Centrifuge

Standards

Standard Company Catolog # Stock Concentration Stock Diluent
Digoxin Sigma D6003 500µg/mL DMF/MeOH
Oleandrin(IS) Sigma 06069 500µg/mL MeOH
Dihydrodigoxin Custom synthesized NA 500µg/mL DMF/MeOH

Instrument

  • Pump: Shimadzu LC-30 AD
  • Injector: Shimadzu SIL- 30AC set at 4°C
  • Detector: LC/MS/MS API Qtrap 6500
  • Column: ODS Hypersil 4.6 x 100 mm, 5 µm particle size (or equivalent)

Run Conditions

  • Mobile phase: 65/35 (v/v) MeOH:water
  • Column Temperature: 50°C
  • Inection Volume: 10-20µL
  • Run Time 5.0min
Metabolite Elution Time(min)
Digoxin 2.0
Dihydrodigoxin 2.1
Oleandrin(IS) 3.6
  • Sample Inlet: Turbo Spray IonDrive
  • Mass scanning mode: Negative MRM (Multiple Reaction Monitor)
Metabolite Q1 (m/z) Q3 (m/z)
Digoxin 779.4 475.2
Dihydrodigoxin 781.4 521.1
Oleandrin(IS) 575.2 531.1
  • Ionization: ESI/Negative Ionization
Parameter Setting
Duration 5.0min
Cycle Time 0.615sec
Cycles 488
Scan Time 200 msec
Scan Type Negative MRM
Resolution Q1 unit
Resolution Q3 unit
Intensity Threshold 0.00 cps
Smart Setting off
Settling Time 0.00 msec
MR Pause 5.00 msec
MCA No
GS1 55
GS2 55
CUR 20
Temperature 600°C
CAD High (N2 = 99.999%)
Step Size 0.00 amu
DP -40
EP -10.00
CE Digoxin -59
CE Dihydrodigoxin -56
CE Oleandrin (IS) -45
CXP 12
IS 4500
Weighted linear regression 1/x2

Preparation of Standards

Standards are prepared by mixing 45µL H2O with 5µL serum and the volumes below:

Standard 50% ACN (µL) Dig Working Solution (ng/mL) Working Solution (µL) Stanrdard Curve Concentration (ng/ml)
00 20 0 0 0
0 20 0 0 0
1 18 2.5 2 0.1
2 15 2.5 5 0.25
3 10 2.5 10 0.5
4 0 2.5 20 1
5 18 50 2 2
6 15 50 5 5
7 10 50 10 10
8 0 50 20 20

QC samples are prepared according to the volumes below:

QC Sample 50% ACN (µL) Dig Working solution (ng/mL) Volume Spiked (µL) Nominal Concentration)
Low 14 2.50 6 0.75
Medium 18 50 2 5.0
High 4 50 16 40

Extraction

  • Mix 45µL H2O with 5µL sample in serum, and 20 µL of 50% acetonitrile/water
  • Add 100µL of internal standard (10ng/mL oleandrin) and mix
  • Add 1 mL of 1:1 methyl t-butyl ether: ethyl acetate, and mix well by vortex
  • Centrifuge at 3000rpm for 10 min
  • Freeze the aqueous layer in dry ice/alcohol bath
  • Transfer organic phase to new 16x48mm tube
  • Evaporate in Turbovap under nitrogen (10psi) at RT for ~30 min or until dry
  • Reconstitute in 65:35 MeOH:H2O and votex well
  • Transfer to autosampler vials and centrifuge for 6 min at 10,000rpm