Validation Of An LC/MS/MS Assay For Tenofovir In Human Serum

SHEELA DAS, Ph.D.
BIOANALYTICAL SYSTEMS, INC.
WEST LAFAYETTE, IN 47906

TENOFOVIR - HIV REVERSE TRANSCRIPTASE INHIBITOR

  • Tenofovir disoproxil fumarate (prodrug)
  • Prodrug requires initial diester hydrolysis for conversion to tenofovir and subsequent phosphorylation by cellular enzymes to form tenofovir diphosphate
  • Tenofovir diphosphate competes with natural substrate deoxyadenosine 5’- triphosphate to inhibit activity of HIV reverse transcriptase and causes DNA chain termination

STRUCTURES OF PRODRUG

TENOFOVIR DISOPROXIL FUMARATE

METHOD DEVELOPMENT CONSIDERATIONS

  • Solubility and stability of analyte stock solution
  • No stable label Internal Standard available. Therefore, important to choose an ISTD that had similar structural chemistry
  • Identifying analytical column that gave good retention and ruggedness. In general most columns used gave poor analyte retention and reproducibility
  • Identifying extraction chemistry that allowed for selective isolation of analyte of interest

Stock Solution

  • Common solvents like methanol, ACN tried unsuccessfully
  • Finally, methanol with 1% ammonium hydroxide was used

Selection of Internal Standard

2'-Deoxyadenosine-5'-monophosphoric acid, monohydrate was chosen because of structural similarities

Identifying an Analytical Column

Column Type Mobile Phase Analyte RT
Betasil C 18 10% acetonitrile with 20mM ammonium formate Not retained
Xterra C 18 10% acetonitrile with 20mM ammonium formate 2.72 min. Column lost peak shape within 50 injections
Ultra IBD 20% acetonitrile with 0.1% formic acid 3.72 min. Was able to run over 200-300 extracts on column without loosing peak shape

Optimizing Extraction Chemistry

Variable Conclusion
Protein Precipitation Ion suppression, therefore aborted.
SPE , anion exchange chemistry Works well, no ion suppression.
Amount of Buffer and pH 200 μL and 400μL of pH 10 and 12 evaluated. Optimized at 400μL of pH 12.
Amount of elution solution 200 μL and 400μL of 2% formic acid evaluated. Optimized at 400μL.

Final Optimized Conditions

  • Column: Ultra IBD 100 x 4.6mm, 5μm
  • SPE using Waters Oasis MAX(30mg)
  • 50 mM Sodium Carbonate (pH12.0) Buffer
  • Elution Solution: 2% formic acid in methanol
  • Mobile Phase: 20% ACN/ 0.1% formic acid

General Assay Procedure - Automation

  • Load Samples onto 96-well plate
  • Add ISTD in buffer
  • Condition plate with methanol and water
  • Load samples
  • Rinse plate with water and methanol
  • Elute with acidic solvent
  • Evaporate to dryness
  • Reconstitute
  • Inject on LC/MS/MS

Assay Specifics

  • Sample Volume: 200 μL
  • Sample Preparation: SPE
  • Validated Range: 5.00 - 500 ng/mL
  • Column: Ultra IBD
  • Mobile Phase: 20% ACN/ 0.1% formic acid
  • Regression: Linear 1/x
  • Detection: LC/MS/MS

Chromatogram of a Extracted Low Calibrator

Typical Calibration Curve

Calibration Standard Statistics (ng/mL)

Nominal Concentration 5.00 10.0 25.0 50.0 100 250 500
Average Concentration 4.55 9.88 25.2 51.6 105 252 491
Standard Deviation 0.236 0.804 1.03 3.03 5.86 13.1 15.2
Precision (%) 5.2% 8.1% 4.1% 5.9% 5.6% 5.2% 3.1%
% Bias -9.0% -1.2% 0.8% 3.2% 4.8% 0.7% -1.8%
N 6 8 8 8 8 8 7

Inter-Assay Quality Control Sample Statistics

Nominal Concentration 400 240 15.0 5.00
Average Concentration 407 244 15.1 4.51
Standard Deviation 17.1 14.4 1.01 0.268
Precision (%) 4.2% 5.9% 6.7% 5.9%
% Bias 1.8% 1.5% 0.7% -9.7%
N 20 20 20 17

Assay Performance over 50 batches

Summary of Validation Results

  • Sample Matrix: Human serum
  • Instrumental Technique: LC/MS/MS
  • Sample Volume: 200 μL
  • Calibration Standards: 5 - 500 ng/mL
  • Regression: Linear 1/x. Quantitation by peak area ratio.
  • Quality control samples: 400 ng/mL, 240 ng/mL, 15.0 ng/mL
  • Freeze/thaw stability: 4 cycles at ~ -20oC
  • Room temperature stability in matrix: Demonstrated ~ 28 hours
  • Processed extract stability: Demonstrated ~ 47 hours at room temperature
  • Heat Treatment Stability: At least 40 minutes at ~ 56°C
  • Stock Solution Stability: At least 51 days at ~ -20oC
  • Long term stability in frozen matrix: At least 30 days at ~ -20ºC

Conclusions

  • Tenofovir a nucleotide reverse transcriptase inhibitor is assayed by reverse phase HPLC using an isocratic mobile phase
  • Assay is rugged, sensitive, and will aid in the pharmacokinetic measurements of Tenofovir