Analytical Considerations

Equilibrium dialysis technique was used by several investigators to estimate free lidocaine concentration. Routledge et al. subjected two 1 ml aliquots of plasma to equilibrium dialysis using a Teflon equilibrium dialysis cell. The dialysis was performed against Sorenson's phosphate buffer (containing 0.5% w/v sodium chloride), and the pH was adjusted to 7.4 to which lidocaine hydrochloride was added (3 ^g/mL buffer). This concentration was achieved by adding unlabeled lidocaine hydrochloride (2.8 ^g/mL) to radioactive 14C lidocaine (200ng/mL). The buffer and plasma compartment were separated by a Spectrapor dialysis membrane with a molecular weight cut-off range of 12,000-14,000, and the cells were rotated in a water bath for 3h at 37°C. The authors demonstrated that equilibrium was achieved in 3h and the binding was similar in heparinized plasma, citrated plasma, and serum. After dialysis, 300 ^l of aliquots were withdrawn from each side of the cells, scintillation fluid was added, and the radioactivity was measured. Quench correction was made

Table 4

Technical Aspects of Monitoring Free Drug Concentration

Table 4

Technical Aspects of Monitoring Free Drug Concentration

Free Drug

Separation from Bound

Analytical Technique

Phenytoin

Ultrafiltration

Immunoassay

Carbamazepine

Ultrafiltration

Immunoassay

Valproic Acid

Ultrafiltration

Immunoassay

Digoxin

Ultrafiltration

Immunoassay3

Lidocaine

Ultrafiltration13

Immunoassay

Quinidine

Ultrafiltrationb

Immunoassay

Cyclosporine

Equilibrium dialysis

HPLC/MS

Tacrolimus

Equilibrium dialysis

HPLC/MS

Mycophenolic Acid

Ultrafiltration

Immunoassay/ HPLC

Indinavir

Ultrafiltration

LC/MS

Amprenavir

Ultrafiltration/

HPLC/UV

equilibrium dialysis

HPLC, high-performance liquid chromatography; UV, ultraviolet. a Because free digoxin is almost 75% of total digoxin concentration, commercially available immunoassays usually have enough sensitivity for accurate determination of free digoxin. b Methods may also include equilibrium dialysis.

HPLC, high-performance liquid chromatography; UV, ultraviolet. a Because free digoxin is almost 75% of total digoxin concentration, commercially available immunoassays usually have enough sensitivity for accurate determination of free digoxin. b Methods may also include equilibrium dialysis.

by the external standard ratio method, and the percentage of unbound drug in plasma was calculated as the ratio of the absolute disintegration rates in buffer and plasma multiplied by 100 (7). Other studies published later also used equilibrium dialysis for estimating unbound lidocaine concentration (8,10).

Edwards et al. studied protein binding of quinidine in human plasma. The quinidine Fu was two- to threefold higher when blood was collected in evacuated blood collection glass syringes. Other factors that affect protein binding of quinidine include addition of heparin in vitro, condition of equilibrium dialysis, and the presence of dihydroquinidine, which may be a common impurity in quinidine preparation. The authors subjected 400 ^l of phosphate buffer solution (pH 7.4, 0.134 M) to equilibrium dialysis against an equal volume of serum in plexiglass cells for 5 h at 37°C. Postdialysis, quinidine concentrations on each side of the dialysis membrane were determined by using liquid scintillation counting (13).

McCollam et al. used ultrafiltration technique and fluorescence polarization immunoassay (FPIA) for determination of unbound concentration of lidocaine and quinidine. For this purpose, serum pH was adjusted to 7.4 ± 0.5 by bubbling carbon dioxide through 1 ml of the specimen. Then, ultrafiltration was performed to separate bound fraction from Fu using a Centricon-10-ultrafiltration device (Amicon, Baverly, MA) at 2500 x g for 45 min. This ultrafiltration device has a molecular weight cut-off of 10,000, which prevents a1-acid glycoprotein to pass through the column. The FPIA assays were performed by using a TDx analyzer (Abbott Laboratories, Abbot Park, IL) (12). Analytical conditions for monitoring free drug concentrations are summarized in Table 4.

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