Creatinine is produced continuously by muscle metabolism and excreted in urine at predictable concentrations, which makes it the primary marker in specimen validity testing. Certified laboratories measure it on every specimen: 20 mg/dL or above is normal; 2–20 mg/dL combined with low specific gravity classifies the specimen as dilute (triggering negative-dilute handling); and below 2 mg/dL — a level incompatible with human physiology — combined with out-of-range specific gravity classifies it as substituted, which DOT treats as a refusal to test. Because creatinine varies with hydration, muscle mass, and diet, the thresholds are set conservatively; the marker exists to catch flushing and swapping, not normal biological variation.