THERAPEUTIC DRUG MONITORING (TDM) & VALIDATION

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
Linearity of Tacrolimus between 1 - 160 ng/ml
S/N Ratios at 1 ng/L for Tacrolimus
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
Linearity of Cyclosporine between 15 - 2000 ng/mL
Chromatograms of Cyclosporine
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
Linearity of METHOTREXATE between 0.5 - 80 ng/mL
Chromatograms of extracted METHOTREXATE samples 20 & 0.5 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
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).
  • CollectionRoutine 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
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
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)