Myoglobin determination by high-performance liquid chromatography.

Urine and serum myoglobin have been separated on an anion-exchange column, packed by the slurry technique. Urine or serum was injected directly into the column and eluted isocratically with Tris buffer. Freshly prepared myoglobin from human muscle gives two peaks in the chromatogram. Upon storage, one peak slowly decreases while the other increases in size and has the same capacity factor as horse myoglobin. In myoglobinuria, the latter peak is usually detected in urine, while in serum both peaks are detected. For routine assays, horse myoglobin is used as a standard. The minimum detectable level is 2 mg/l. Urine myoglobin from patients with myoglobinuria ranged from 20 to 3000 mg/l, while normal subjects had undetectable levels. Patients with myoglobinuria also had detectable levels of myoglobin in their serum. Urine myoglobin was found to be unstable; it should be analyzed immediately. Although the present method is not sensitive enough to detect myoglobin in the urine of normal subjects, it is clinically useful for confirming and determining myoglobin in patients with myoglobinuria. It has the advantage of speed and simplicity. Using more sensitive detectors would enhance the usefulness of this method.