Therapeutic Drug Monitoring and Overdose Testing

A drug qualifies for therapeutic drug monitoring when its concentration correlates with effect and a measurement can guide dosing. That condition covers aminoglycosides, cardioactive drugs, anticonvulsants, lithium, tricyclic antidepressants, and immunosuppressants. Warfarin is monitored by PT/INR, insulin by glucose metrics, and opioids chiefly by clinical response and toxicity, with routine serum concentrations used only in selected contexts. TDM results carry meaning only against the exact dose, collection time, regimen, indication, and protocol that produced them.¹

Pharmacokinetic principles

Absorption and bioavailability. Oral absorption depends on dissolution, GI solubility, and passive diffusion. The nonionized fraction generally crosses lipid membranes more readily, but most orally administered drugs are absorbed primarily in the small intestine because of its large surface area and blood flow. IV administration bypasses absorption entirely and delivers the highest, most predictable bioavailability. Distribution describes movement between blood and tissue. The volume of distribution, Vd = amount of drug in the body divided by plasma concentration, is large for lipophilic drugs that partition into fat and nerve tissue and small for drugs that stay confined to plasma, such as ionized or highly protein-bound compounds. After an IV bolus, Vd may be estimated from dose and the extrapolated initial concentration. Only the free, unbound drug fraction is pharmacologically active; protein-binding changes from inflammation, malignancy, pregnancy, or hepatic and renal disease can shift free fraction and produce toxicity or treatment failure at an unchanged total concentration.¹

Metabolism. The hepatic mixed-function oxidase system, MFO/CYP450, converts hydrophobic xenobiotics to water-soluble excretable products. Phase I reactions oxidize, reduce, or hydrolyze a xenobiotic to introduce or expose a functional group. Phase II reactions conjugate the parent or Phase I product, commonly by glucuronidation, sulfation, acetylation, glutathione conjugation, or amino-acid conjugation. MFO induction shortens drug half-life through faster clearance; hepatic disease such as cirrhosis lengthens it. The first-pass effect describes hepatic metabolism of an orally absorbed drug before it reaches systemic circulation.

Elimination. Most drugs follow first-order kinetics, an exponential constant-fractional decline:

C(t) = C₀ · e^(−kt), and t½ = 0.693 / k

where k is the elimination rate constant and t½ the half-life. Approximately five half-lives commonly approximates steady state on a fixed regimen, but it is not an absolute minimum wait for meaningful TDM: earlier levels may guide loading, suspected toxicity, organ dysfunction, or Bayesian and AUC dosing. Interpret every result against the exact dose, collection time, regimen, indication, and protocol.¹

Worked example. A vancomycin concentration is 30 µg/mL at 09:00 and 20 µg/mL at 15:00, six hours later. The elimination constant is k = −ln(20/30)/6 = 0.0676/h, giving an approximate half-life of 0.693/0.0676 = 10.3 hours. If the same first-order elimination continues for another six hours, the predicted 21:00 concentration is 20 × e^(−0.0676 × 6) = 13.3 µg/mL. This estimates the individual patient’s elimination kinetics; actual vancomycin dosing follows protocol-specific AUC-guided interpretation under the 2020 ASHP/IDSA consensus, which targets AUC/MIC 400-600 for serious MRSA infections rather than a trough-only target.²

Pharmacodynamics. The dose or concentration-response curve relates drug level to effect. EC₅₀ is the concentration producing 50% of maximal effect. ED₅₀, TD₅₀, and LD₅₀ are doses producing the specified effect in 50% of a population, and the conventional therapeutic index is TD₅₀/ED₅₀, or LD₅₀/ED₅₀ in preclinical use.

Specimen timing is the major preanalytic variable. Document the dose, infusion, and collection times. Draw a trough immediately before the next dose only when the drug-specific protocol calls for one; peak or post-dose timing varies with formulation, infusion duration, and regimen. Aminoglycoside sampling differs between conventional and extended-interval dosing and must follow the local protocol. Clinically useful levels may be collected before steady state. EDTA, citrate, and oxalate specimens are generally unacceptable for TDM because calcium-binding anticoagulants alter drug distribution and binding, with the recognized exception of EDTA whole blood for immunosuppressant monitoring.

Monitored drugs by class

Therapeutic ranges are method-, assay-, and protocol-dependent. A transplant program validates its target range against one specific assay, and serial results for one patient stay on that platform.

Test procedures and interferences

Immunoassay principle. Most therapeutic drugs are quantified by competitive immunoassay, where drug in the specimen competes with a fixed amount of labeled drug for a limited quantity of drug-specific antibody, in enzyme, chemiluminescent, turbidimetric or nephelometric-inhibition, or fluorescence-polarization format. Signal typically falls as drug concentration rises; some homogeneous enzyme-multiplied formats instead show a rising signal. The immunology module owns the general format mechanics. LC-MS/MS serves as the reference method and, for several drug classes, the routine method of choice wherever antibody cross-reactivity is clinically unacceptable, most notably immunosuppressants, because it resolves parent drug from metabolites and structurally related compounds by mass.¹

Cross-reactivity. Antibody-based assays cannot always distinguish parent drug from an active or inactive metabolite, or from a structurally related compound:

• Digoxin immunoassays cross-react with endogenous digoxin-like immunoreactive substances, DLIS, which are elevated in neonates, pregnancy, and renal or hepatic failure, producing a falsely elevated result in a patient not taking digoxin.
• Digoxin-specific antibody Fab fragments given as antidote bind and inactivate circulating digoxin but leave it immunoreactive. A routine total-digoxin immunoassay drawn afterward can read markedly and misleadingly high for several days, longer with renal impairment, and cannot guide dosing until the Fab has cleared or a validated free-digoxin method, using ultrafiltration or equilibrium dialysis, is used.
• Immunosuppressant metabolites of tacrolimus, cyclosporine, and sirolimus cross-react with the parent-drug antibody to a degree that varies by manufacturer, so an immunoassay result can run higher than the true parent-drug concentration by LC-MS/MS; the discrepancy must be reconciled against the assay used to validate the transplant program’s target range.

Specimen requirements. Tacrolimus, cyclosporine, and sirolimus distribute heavily into erythrocytes and require whole blood, EDTA, as the specimen; serum or plasma substantially underestimates the circulating drug. Other TDM specimens are assay-specific; follow the performing laboratory’s requirements. In particular, collect lithium in serum and never in lithium-heparin plasma.

Timing documentation. Record both the exact dose or infusion time and the exact specimen collection time on every TDM request; a result cannot be interpreted against a pharmacokinetic target without both. Draw a trough specimen immediately before the next scheduled dose when the protocol calls for a trough. Peak timing is drug- and route-specific, shifted by formulation, infusion duration, and regimen, and must follow the specific drug and regimen protocol.

Collection-device interference. Gel-barrier separator tubes can adsorb lipophilic drugs onto the gel over time, with phenytoin the classic example, falsely lowering the measured concentration. TDM specimens for such drugs are collected in a plain, non-gel tube, or separated promptly to limit gel contact.

Free versus total drug. For highly protein-bound drugs such as phenytoin and valproic acid, only the free fraction is pharmacologically active. Hypoalbuminemia, uremia, where retained organic acids displace drug from binding sites, and pregnancy, which alters protein binding and volume of distribution, can lower the bound fraction and produce toxicity or altered response at a total concentration that still looks therapeutic; free-drug measurement should be requested in these settings. Albumin-correction formulas apply to total drug and are less reliable in uremia.³

Therapeutic-drug overdose

Salicylates, including aspirin. Salicylates inhibit cyclooxygenase, reducing thromboxane and prostaglandin synthesis, which causes platelet dysfunction and GI injury. They are also epidemiologically linked to Reye syndrome in children with viral illness. Acute overdose produces a classic mixed acid-base picture: salicylate is an acid and contributes to metabolic acidosis while directly stimulating the respiratory center to cause respiratory alkalosis. Krebs-cycle inhibition produces excess lactate, and lipolysis drives ketogenesis, with nonrespiratory, metabolic, acidosis becoming the dominant late finding. Interpret serial concentrations with acuity, acute versus chronic, pH, symptoms, renal function, and the clinical trajectory. Chronic toxicity can be severe at substantially lower concentrations than acute toxicity, and treatment is never withheld because a concentration sits below 50 mg/dL. The Trinder reaction, a ferric nitrate colorimetric complex, is a common screening method; enzymatic and chromatographic methods offer higher specificity. Obtain serial salicylate concentrations until clearly declining and ongoing absorption is unlikely. A falling serum level can be misleading because acidemia increases CNS entry.⁴

Acetaminophen. Acetaminophen is safe at therapeutic doses. Overdose depletes hepatic glutathione, which is needed to conjugate a reactive MFO-pathway intermediate, so the intermediate accumulates and causes hepatocyte necrosis. Early symptoms and laboratory results can be deceptively normal, but AST and ALT usually begin rising within 24 to 72 hours and may peak around 72 to 96 hours. After a single acute ingestion at a known time, a serum concentration drawn at least 4 hours after ingestion is interpreted on the Rumack-Matthew nomogram, where the US-Canada treatment line crosses 150 µg/mL at 4 hours; concentrations on or above the treatment line at 4 hours trigger antidotal treatment. Values before 4 hours cannot be risk-stratified on the nomogram. Unknown-time, repeated, or staggered ingestions follow a different interpretive approach: the 2023 US and Canada consensus starts treatment for repeated supratherapeutic ingestion when the transaminases are abnormal or the concentration exceeds 20 µg/mL. Routine clinical quantification for acetaminophen varies by platform; immunoassay and chromatographic methods are both in use, and chromatography provides the reference capability.⁴

Drugs of abuse

Urine screening by immunoassay followed by confirmatory chromatography or mass spectrometry is the standard two-tiered approach, and a positive screen is presumptive only. Screening detects recent use and cannot distinguish a single exposure from chronic use. Documented chain of custody is required whenever results may be used for employment, legal, or forensic purposes.⁵

Detection windows depend on the cutoff, the assay, dose, use pattern, metabolism, and individual variation; the table gives the ranges commonly cited for standard urine immunoassay screening: