RATL is the only institutional lab to be assessed, audited and approved for TDM by the ISO 17025 / NABL accreditation authority in India.
TDM has been validated with a high degree of accuracy, precision, repeatability and sensitivity with lowest detection levels (0.1ng/ml) by the gold-standard method - LCMSMS using a high-end Waters XEVO-TQD-UPLC system, one of the most advanced in the world today.
TDM is carried out across multiple categories:
- Immunosuppressants - Tacrolimus, Cyclosporine A, Methotrexate
- Antibiotics - Gentamycin, Vancomycin, Amikacin
COMPARATIVE FEATURES OF IMMUNOASSAYS vs. LC-MS-MS
IMMUNOASSAYS
- Immunoassay is known to be nonspecific and suffers from interferences.
- Cross-reactions between drugs and metabolites may result in overestimation of the drug concentration with unacceptable bias. The presence of auto anti-bodies and/or heterophile antibodies, falsely elevate results in immunoassays.
- The degree of cross reactivity or interference metabolites was greater in
- They are high in cost and are not able to assay multiple drugs simultaneously.
- The LLOQ of the IA techniques is often not low enough.
- Lack of result comparability occurs between different assays and often between the same assays performed in different laboratories.
LC-MS-MS
- LC-MS/MS is considered the most sensitive, specific and precise technology for monitoring immunosuppressants.
- Increased sensitivity and analytical specificity is superior to that of immunoassays.
- Metabolite cross-reactivity is not an issue with LC–MS/MS. LC/MS/MS approach is more suitable for simultaneous determination of multiple analytes/metabolites.
- LC/MS/MS methods are more robust while performing lab-to-lab and assay-to-assay comparisons with other MS/MS.
- The analytical “gold standard,” delivering high quality and specificity, particularly for small molecule analytes including drugs of abuse, therapeutic drugs, immuno-suppressants, steroids, and vitamin D metabolites.
IMMUNOSUPPRESSANTS
TACROLIMUS
- Linearity: 1 - 160 ng/mL
- LoQ: 1 ng/mL
- Method of Assay: LC-MS-MS
Linearity of Tacrolimus between 1 - 160 ng/ml
S/N Ratios at 1 ng/L for Tacrolimus
SAMPLE INFORMATION
- Sampling time: Pre-dose (trough) levels (Draw blood immediately before a schedule dose)
- Specimen : 3ml whole blood
- Container : Lavender top (EDTA) tube
- Collection : Routine venipuncture
- Special processing : Do not centrifuge. Specimen should remain as whole blood in original collection container.
- Storage/Transport temperature: Refrigerated temperature
- Unacceptable conditions : Serum, plasma, clotted specimens or specimens left at room temperature for longer than 24 hours
- Stability : Ambient: 24 hours; refrigerated: 2 weeks; frozen: 2 months
- Additional information : Therapeutic range applies to trough specimens drawn immediately prior to AM dose
PHARMACOKINETICS
- Time to peak : 1-3 hrs.
- Route of elimination : Hepatic metabolism <1% excreted renally
- Elimination half-life : 10-20 hr. (range 4-53)
- Time to steady state : 2-5 days
- Protein binding : >98%
- Target range : Varies with sample time, transplant type and time after transplantation. Typically 15 µg/L (18.2 nmol/L) following kidney transplantation reducing to 5-10µg/L(6.1 – 12.2 nmol/L)
CYCLOSPORINE
- Linearity: 15 - 2000 ng/mL
- LoQ: 15 ng/mL
- Method of Assay: LC-MS-MS
Linearity of Cyclosporine between 15 - 2000 ng/mL
Chromatograms of Cyclosporine
SAMPLE INFORMATION
- Sampling time: Trough(1 hour before next dose)or Peak (2 hours post dose)
- Specimen: 3ml whole blood
- Container: Lavender top (EDTA) tube
- Collection: Routine venipuncture. Do not draw specimen from indwelling catheter which has been used to administer Cyclosporine as it is adsorbed by the catheter and elevates blood values significantly.
- Special processing : Do not centrifuge. Specimen should remain as whole blood in original collection container.
- Storage/Transport temperature: Refrigerated temperature
- Unacceptable conditions: Serum, plasma, clotted specimens, specimens left at room temperature for longer than 24 hours
- Stability : Ambient: 24 hours; refrigerated: 2 weeks; frozen: 2 months
- Additional information: Therapeutic range applies to trough specimens drawn immediately prior to AM dose
PHARMACOKINETICS
- Time to peak: 1-6 hrs.
- Route of elimination: Hepatic metabolism <1% excreted renally
- Elimination half-life: 12 hr (range 4-53)
- Time to steady state: 2-6 days
- Protein binding: >98%
- Target range: Varies widely with sample time, transplant type and time after transplantation
METHOTREXATE
- Linearity: 0.5 - 80 ng/mL
- LoQ: 0.5 ng/mL
- Method of Assay: LC-MS-MS
Linearity of METHOTREXATE between 0.5 - 80 ng/mL
Chromatograms of extracted METHOTREXATE samples 20 & 0.5 ng/mL
SAMPLE INFORMATION
- Sampling time: As required by protocol, often 24, 48 & (if necessary) 72 hours post single high-dose therapy.
- Specimen: 2ml serum. Protect from light during collection, storage, and shipment.
- Container: Red top (no gel). Also acceptable: Green (sodium heparin), lavender (EDTA), pink (K2EDTA), or gray (sodium fluoride/potassium oxalate).
- Collection: Routine venipuncture. Separate serum from cells within 2 hours of collection.
- Special processing : Allow to clot. Centrifuge specimen and separate serum/plasma within 2 hours of collection, aliquot into amber plastic vial. Store at refrigerated temperatures.
- Storage/Transport temperature: Frozen. Wrap collected specimens in foil to protect from light during transport to the laboratory.
- Unacceptable conditions : Serum separator tubes. Specimens not protected from light.
- Stability: After separation from cells, ambient: 2hrs; refrigerated: 24hrs; frozen: 1 month
PHARMACOKINETICS
- Time to peak: 1hr for low-dose oral therapy
- Route of elimination: Predominently renal excretion, 90% in high dose regimes
- Elimination half-life: 5-9 hr (less if urine alkalinized)
- Time to steady state: 1-2 days of chronic low dosing
- Protein binding: 50%
- Target range: <1 µmol/L (<450 µg/L) 48 h post high-dose therapy or according to protocol. (The convention is to use molar SI rather than mass SI units for methotrexate concentrations)
AMINOGLYCOSIDE ANTIBIOTICS
AMIKACIN
- Linearity: 1 - 100 μg/mL
- LoQ: 1 μg/mL
- Method of Assay: LC-MS-MS
Linearity of AMIKACIN between 1 - 160 ng/mL and chromatogram of AMIKACIN
SAMPLE INFORMATION
- Sampling time: Peak (only used on divided-dose regimes): 1 hour post dose (30-60 min after IV infusion complete, 60 min after IM).
- Trough: Immediately before next dose.
- Specimen: 5ml serum
- Container: Red top (no gel), also acceptable: Green (sodium heparin).
- Collection: Routine venipuncture. Separate serum or plasma from cells within 1 hour of collection.
- Special processing: Centrifuge specimen, separate serum/plasma within 1 hour of collection, aliquot into plastic vial. Store at refrigerated temperature.
- Storage/Transport temperature: Refrigerated, frozen (if transport is prolonged)
- Unacceptable conditions: Specimens collected in citrate or oxalate/fluoride, specimens exposed to repeated freeze/thaw cycles.
- Stability: After separation from cells, ambient: 4 hours; refrigerated: 1 week; frozen: 2 weeks (avoid repeated freeze/thaw cycles
PHARMACOKINETICS
- Time to peak: 1 hr
- Route of elimination: >90% excreted renally
- Elimination half-life: 2-3 hr with normal renal function
- Time to steady state: 10-15 h with normal renal function
- Protein binding: <10%
- Target range: Trough: <10mgOn once-daily dosing, target is a trough concentration of <5 mg Peak: 20-30 mg/L. Once daily extended-dose regimens seek local advice
GENTAMICIN
- Linearity: 1 - 100 μg/mL
- LoQ: 1 μg/mL
- Method of Assay: LC-MS-MS
Linearity of GENTAMICIN between 1 - 160 ng/mL and chromatogram of GENTAMICIN
SAMPLE INFORMATION
- Sampling time: Peak: 1 hour post dose (30-60 min after IV infusion complete).
- Trough: Immediately before next dose.
- Specimen: 5ml serum
- Collection: Routine venipuncture.
- Special processing: Allow specimen to clot completely. Centrifuge specimen, separate serum/plasma from cells, transfer into plastic vial.
- Storage/Transport temperature:Refrigerated. frozen(if transport is prolonged)
- Unacceptable conditions : Separator tubes, plasma collected in citrate or oxalate/fluoride tubes or EDTA, hemolyzed specimens
- Stability: After separation from cells, ambient: 24 hours; refrigerated: 1 week; frozen: 2 weeks (avoid repeated freeze/thaw cycles)
PHARMACOKINETICS
- Time to peak: 1 hr
- Route of elimination: >90% excreted renally
- Elimination half-life: 2-3 hr with normal renal function
- Time to steady state: 10-15 h with normal renal function
- Protein binding: <10%
- Target range: Once daily extended-dose regimens (seek local advice for specific regime), multiple dose regimens: trough: <2 mg/L (<1 in cases with endocarditis)
VANCOMYCIN
- Linearity: 1 - 100 µg/mL
- LoQ: 1 µg/mL
- Method of Assay: LC-LC-MS
Linearity of VANCOMYCIN between 1 - 100 ug/mL and chromatogram of VANCOMYCIN
SAMPLE INFORMATION
- Sampling time: Peak: 1 hour post dose (30-60 min after IV infusion complete).
- Trough: Immediately before next dose.
- Specimen: 5ml serum
- Container: Red top (no gel), also acceptable - heparinized plasma
- Collection: Routine venipuncture, separate serum or plasma from cells within 2 hours of collection.
- Special processing: Separate serum from cells as soon as possible or within 2 hours of collection. Transfer serum/plasma to a plastic vial.
- Storage/Transport temperature: Frozen.
- Unacceptable conditions: Gray (sodium fluoride/potassium oxalate), lavender (EDTA), blue (sodium citrate), separator tubes, grossly hemolyzed, icteric or lipemic specimens
- Stability: After separation from cells, ambient: 8 hours; refrigerated: 1 week; frozen: 2 weeks
PHARMACOKINETICS
- Time to peak: 1 hr
- Route of elimination: >80% excreted renally
- Elimination half-life: 4-7 hr with normal renal function(longer in the elderly)
- Time to steady state: 20-35 h with normal renal function
- Protein binding: <10%
- Target range: Trough: 5-15 mg/L (3-7 µmol/L), peak: 20-40 mg/L (14-28 µmol/L) (Note: peak levels are no longer routinely recommended.Therapeutic levels of vancomycin must be individually established based on patient differences and bacterial susceptibility)