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Important scientific principles of pain medicine pharmacology affect urine drug testing (UDT). This paper reviews sources of variability in pharmacokinetics, pharmacodynamics, pharmacogenetics, and issues relating to the collection, handling, and assay of urine and how these factors may affect test interpretation and application.Articles concerning the pharmacokinetics, pharmacodynamics, and pharmacogenetics of opioids are reviewed and interpreted for pain clinicians who treat patients with chronic opioid therapy. These data are applied to the use of UDT.Intraindividual and interindividual variability in drug metabolizing enzyme activity due to genetic polymorphisms or environmental effects can result in day-to-day and patient-to-patient variability in drug exposure. Transporters, also under genetic and environmental control, can play an important role in opioid response and contribute to the significant variability in opioid pharmacokinetics and response. The use of urine creatinine concentration to adjust urine drug concentrations, discussion of UDT assays, and application of UDT in light of an understanding of pharmacokinetics, pharmacodynamics, and pharmacogenetics are reviewed. In addition, the methodology used for testing has an important role in accuracy. Because of these factors, UDT cannot be used to determine patient compliance with a specific opioid dose.UDT, when used with an understanding of the principles of pharmacokinetics, pharmacodynamics, and pharmacogenetics of opioids, can be a useful tool in chronic pain management. Clinicians must keep in mind the limitations, purpose, and value of UDT, and the inability to predict patient compliance with a drug dosage using commercial algorithms.