Monitoring patient compliance and bioavailability of drugs by deproteinizing body fluids

• US 6,790,668 B1
• Filed: 09/07/2000
• Issued: 09/14/2004
• Est. Priority Date: 11/23/1998
• Status: Expired due to Term

• Current Assignee: Fundação Oswaldo Cruz Fiocruz
• Original Assignee: Fundacao Oswaldo Cruz - Fiocruz
• Inventors: Luiza, Vera L., Gemal, André L., Barroso, Eduardo W., Ferreira, Milton F.
• Primary Examiner(s): Ludlow, Jan M.
• Application Number:
  US09/600,594
• Time in Patent Office: 1,468 Days
• Field of Search: 436/175, 436/177, 436/91, 436/92, 436/93, 436/96, 436/98, 436/161, 436/164, 422/61, 422/70, 422/72
• US Class Current: 436/161
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
• CPC Class Codes: G01N 33/9446 Antibacterials
• CPC Class Codes: Y10T 436/141111 Diverse hetero atoms in same or different rings [e.g., alkaloids, opiates, etc.]
• CPC Class Codes: Y10T 436/142222 Hetero-O [e.g., ascorbic acid, etc.]
• CPC Class Codes: Y10T 436/145555 Hetero-N
• CPC Class Codes: Y10T 436/147777 Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

The present invention is a method for drug level detection by using a simplified and effective deproteinizing step from body fluids, such as plasma, blood, urine, saliva, tear fluid, followed by drug extraction and measurement using an accurate technique, such as a colorimetric assay or a High-Performance Liquid Chromatography method. In a particular embodiment, the invention is directed to a method to quantify rifampicin in order to monitor its levels in body fluids and also to a Kit for rifampicin concentration measurement.

Claims

1. Method of monitoring patient compliance and bioavailability of drugs contained in a body fluid consisting essentially of the following steps:

(a) mixing and shaking mechanically the body fluid with a 0.1 M to 5.0 M aqueous zinc sulfate solution to precipitate proteins, and an appropriate solvent to extract the drug during deproteinizing, in a single step, and recovering at least 97% of the drug;

(b) centrifuging the mixture of (a), to obtain the separation of phases;

(c) recovering the supernatant of (b) and measuring the drug concentration in body fluid using a colorimetric assay or a High-Performance Liquid Chromatography method, and

(d) obtaining drug levels down to at least 0.3 μ

g/ml.

2. Method according to claim 1 wherein the concentration of the aqueous zinc sulfate solution ranges from 0.2 M to 1.0 M.

3. Method according to claim 1 or 2 wherein the appropriate solvent is a polar solvent or a non polar solvent or mixtures thereof.

4. Method according to claim 3 wherein the solvent is an organic solvent selected from the group consisting of acetonitrile/2-propanol, benzene, toluene, dichloromethane, chloroform and mixtures thereof.

5. Method according to claim 3 wherein the solvent is selected from the group consisting of water, an alcohol and a mixture thereof.

6. Method according to claim 1 wherein, optionally, an antioxidizing agent is included in step (a).

7. Method according to claim 1 or 2 or 6 wherein the drug is rifampicin.

8. Method according to claim 4 wherein the drug is rifampicin.

9. Method according to claim 1 wherein the drug is selected from the group consisting of an antimonial, an itraconazole, a proteinase and a reverse transcriptase inhibitor.

10. Method of monitoring patient compliance and bioavailability of rifampicin contained in a small amount of a body fluid comprising the following steps:

(a) mixing and shaking mechanically the body fluid with a 0.1 M to 5.0 M aqueous zinc sulfate solution, an organic solvent selected from the group consisting of acetonitrile/2-propanol, benzene, toluene, dichloromethane, chloroform and a mixture thereof, to extract rifampicin and, optionally, an antioxidizing agent to precipitate proteins and strip off bound drug at same time or in a single step;

(b) centrifuging the mixture of (a) to obtain the separation of phases;

(c) recovering the organic phase supernatant of (b) and measuring the drug concentration in said supernatant by using a colorimetric assay or a High-Performance Liquid Chromatography method down to at least 0.3 μ

g/ml.

11. Method according to claim 10 wherein the concentration of the aqueous zinc sulfate solution ranges from 0.2 M to 1.0 M.

12. Method according to claim 10 or 11 wherein the solvent used in step (a) is acetonitrile/2-propanol.

13. Method according to claim 10 wherein said antioxidizing agent is ascorbic acid.

14. Method according to claim 10 wherein the rifampicin concentration is determined through spectrophotometric measurement at 340 nm.

15. Kit for measuring rifampicin concentration in a body fluid containing the following components:

(a) a standard solution of 0.1 M to 5.0 M of aqueous zinc sulfate, optionally, including an antioxidizing agent;

(b) an organic solvent selected from the group consisting of acetonitrile/2-propanol, benzene, toluene, dichloromethane, chloroform and a mixture thereof;

(c) a serum standard containing a know amount of rifampicin to prepare a standard curve for user conditions.

16. Kit according to claim 15 wherein the concentration of the aqueous zinc sulfate solution ranges from 0.2 M to 1.0 M.

17. Kit according to claim 15 wherein said antioxiding agent is ascorbic acid.

18. Kit according to claim 15 wherein the organic solvent is acetonitrile/2-propanol.