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
