Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
2001-08-28
2003-09-09
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S634000, C210S638000, C210S656000, C210S198200, C436S161000
Reexamination Certificate
active
06616846
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to methods and polymeric reagents useful for the extraction and detection of phosphonates present in samples obtained from a variety of environmental and biological sources.
An increasing variety of phosphonic acids and their phosphonate salts have found commercial use in recent years. In the field of agronomy, for instance, N-phosphonomethylglycine, known as glyphosate or Roundup®, has found widespread use as a herbicide, to the extent that its presence in groundwater and in soils has become a matter of environmental concern. Many countries now monitor and regulate the levels at which this herbicide, and its metabolite, aminomethylphosphonic acid (AMP), is present in the environment.
The phosphonates are also becoming widely used in the medical field. Their activity as bone resorption inhibitors, particularly in the form of bisphosphonates, has motivated their use particularly in the treatment of bone disorders such as osteoporosis. Currently, alendronate, etidronate and many other bisphosphonates are approved for commercial use in humans. Other phosphonates have found application as antibiotics, such as fosfomycine, fosfosal, and foscarnet, or as inhibitors of proteolytic enzymes, such as renin and HIV protease. Furthermore, phosphonic acids are found in high concentration in wastewater from textile industries.
There is accordingly a demand for techniques capable of detecting the presence of these and other phosphonates present in samples obtained from different sources.
To provide assays sensitive enough to detect these phosphonates at the very low levels present in most samples, approaches have been developed that utilize a multitude of extraction and enrichment steps to concentrate the analyte, and various chemical treatments to derivatize the analyte, thereby to generate analytical samples that are then subjected to one or more in-line instruments that provide ultrasensitive and either direct or indirect measurement of the phosphonate. U.S. Pat. No. 4,454,043 for instance describes the use of chromatographic columns packed with cation exchange resin, as part of a multi-step procedure for the isolation and detection of glyphosate. Typical of such multi-step procedures is the mass spectrometry-based procedure for glyphosate detection reported by Boyer et al in Anal. Chem., 2000, 72:3826, in which samples are first subjected to column chromatography on ion exchange resins, derivatized with TFAA/NFB, and then subjected to capillary gas chromatography with an ion-trap tandem mass spectrometric detector. Similarly, and as reported by Sparidans et al in J. Chromatog. B., 2000, 738,331, detection of the bisphosphonate known as olpadronate has required a first co-precipitation step of the drug sample with calcium salts, followed by centrifugation and re-dissolving, with each step being repeated at least twice, followed by an ion exchange chromatography step before analysis.
Alternative extraction procedures for phosphonates have been developed more recently, and include the use of immunoaffinity approaches. In WO00/14538, for instance, glyphosate detection is achieved using antibodies raised against a glyphosate conjugate immunogen, in a typical ELISA format.
More suitably, extraction and concentration of such phosphonates, and assays for their detection, would be performed using a rapid and simpler procedure. It is accordingly an object of the present invention to provide an improved process for extracting phosphonates. It is another object of the present invention to provide a polymer useful in such extractions., to permit sensitive and reliable evaluation of samples suspected of containing the phosphonate, whether obtained from human body fluid or from an environmental source.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided an affinity-based method for extracting a phosphonate from aqueous solution, which utilizes a polymer-based zinc ligand as solid phase. More particularly, the present method comprises the step of bringing said sample into contact with a polymeric zinc carboxylate under conditions permitting binding between said phosphonate and said zinc. In another of its aspects, the present method comprises the further steps of treating the polymeric zinc carboxylate to release phosphonate bound thereto, and collecting the released phosphonate. In yet another aspect, the present method entails the further step of analyzing the released phosphonate to detect the level thereof present in the aqueous sample.
Also provided by the present invention, in accordance with another of its aspects, is a polymer useful as solid phase in the extraction of phosphonate, wherein the polymer is the reaction product of zinc methacrylate and a suitable crosslinker. In embodiments, the zinc polymer is provided as packing for use in column chromatography, and the present invention thus also provides a chromatographic column having packed therein a zinc polymer of the present invention.
These and other aspects of the invention are now described in greater detail.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to extraction of phosphonates and phosphonic acids from various sample sources using a zinc polymer as solid phase.
As used herein, the terms “phosphonate” and “phosphonic acid” are used interchangeably with reference to compounds of the formula R—PO
3
═
and O-derivatized forms thereof, and includes the bisphosphonates which typically conform to the general formula
═
O
3
P—R—PO
3
═
, wherein R is selected from a wide variety of radicals. In principle, the present method and polymer are useful to extract any phosphonate that, under binding conditions exploited in the assay, is able to serve as multiple electron pair donor to the immobilized zinc, and is particularly useful to extract phosphonates in which the electron-donating phosphonate oxygen atoms are available for interaction with zinc, as acceptor atom. Non-limiting examples of such phosphonates which can usefully be extracted and detected in accordance with the present methods include the monophosphonates as well as the bisphosphonates, such as glyphosate, its metabolite aminomethylphosphonic acid, and (1-aminobutyl)phosphonic acid, DL-2-amino-3-phosphonopropionic acid (3-phosphono-DL-alanine), DL-2-amino-5-phosphonovaleric acid (DL-5-phosphononorvaline), 4-aminobenzylphosphonic acid, and phenylphosphonic acid, as well as the antibiotics fosfomycin, fosfosal and foscarnet. Examples of bisphosphonates that can useful be extracted and detected by the present method include alendronate, pamidronate, residronate, clodronate, ibandronate, incadronate, neridronate, olpadronate, etidronate, and numerous others. The chemical structures of such compounds are well known, and are reviewed for instance by Fleisch in Endocrine Reviews, 1998, 19(1):80. Common among such phosphonates is the presence in the chemical structure of a zinc-binding oxygen atom, which allows formation of a Zn//O— bond which can be broken by lowering of pH or addition of a chelating agent.
For use as solid phase in the extraction of such phosphonates, and unlike ion exchange resins exploited in other extraction systems, the present invention provides a zinc polymer in which the zinc is presented for binding releasably to the phosphonate to be extracted. The zinc polymer serves as a metal affinity chromatographic polymer, and retains zinc with far greater affinity than the ion exchange resins. The zinc polymer of the present invention is desirably provided as a poly zinc carboxylate, and most desirably as a zinc methacrylate monomer which has been cross-linked using any suitable crosslinking agent. In one embodiment of the present invention, the zinc polymer is the reaction product of zinc methacrylate and ethylene glycol dimethacrylate. In another embodiment of the present invention, the zinc polymer is the reaction product of zinc methacrylate and ethylenebisacrylamide. Alternatively, polymerizing zinc carboxylates other than zinc me
Benrebouh Amina
Idziak Irene
MDS (Canada) Inc.
Rothwell Figg Ernst & Manbeck
Therkorn Ernest G.
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