Researchers Submit Patent Application, “Methods and Apparatus for Analysis of Phase-I and Phase-II Metabolites and Parent Compounds without Hydrolysis”, for Approval
By a News Reporter-Staff News Editor at Life Science Weekly — From Washington, D.C., NewsRx journalists report that a patent application by the inventors HERMAN, Joseph (West Chester, PA); FAIR, Sarah J. (Manchaug, MA); ARGOTI, Dayana (Charlestown, MA), filed on December 21, 2012, was made available online on July 3, 2014 (see also Patents).
No assignee for this patent application has been made.
News editors obtained the following quote from the background information supplied by the inventors: “Liquid chromatography mass spectrometry (‘LCMS’) is a powerful analyte detection and measurement technique that has become the preferred method of detecting small molecule, amino acid, protein, peptide, nucleic acid, lipid, and carbohydrate analytes to a high accuracy for diagnostic purposes. The chromatographic separation process relies on the fact that a number of component solute molecules in a flowing stream of a fluid percolated through a packed bed of particles, known as the stationary phase, can be efficiently separated from one another. Generally, separation in liquid chromatography is achieved in a column by selective distribution of the sample molecules between a stationary phase and a mobile phase. The individual sample components are separated because each component has a different affinity for the stationary phase, leading to a different rate of migration for each component and as different exit time for each component emerging from the column. The separation efficiency is determined by the amount of spreading of the solute band as it traverses the bed or column.
“Reversed-phase liquid chromatography (RPLC) is widely used as a mode of separation in chromatographic systems. In the RPLC technique, the solvent(s) employed in the mobile phase is/are more polar than the stationary phase, whereas the reverse situation is true in conventional (normal phase) chromatography. The mobile phase solvents typically employed in reversed phase liquid chromatography systems comprise water and one or more water-miscible organic modifiers, for example, acetonitrile or methanol. Analyte species of-interest typically form a solution with the mobile phase. The RP-HPLC stationary phase is usually highly hydrophobic or non-polar. The affinity of a chemical species for a stationary phase, which affects the rate at which the particular species in a flowing mobile phase passes through the stationary phase, results primarily from interaction of the species with chemical groups present on the stationary phase. These chemical groups may be provided on the stationary phase by reacting a surface-modifying reagent with a substrate, such as a silica substrate. Surface-modifying agents may thus be employed to adsorb specific chemical groups onto the stationary phase. Conventional reversed-phase liquid chromatography uses 1.5-10 .mu.m spherical silica beads that have been modified by covalent attachment of hydrocarbon chains including 4, 8, or 18 carbon atoms to provide a non-polar surface.
“Clinical laboratories routinely measure the concentrations of drug compounds (including pharmaceutical compounds as well as other natural and synthetic drugs of abuse) in human-provided body fluids. These COM pounds generally cart low molecular weight molecules that are very hydrophilic and difficult to retain on most reversed phase LC systems. The metabolites are even more hydrophilic and are even harder to retain on reverse phase LC systems. Alternative HPLC systems, such as those employing HILIC columns, retain the metabolites but not all of the parent compounds. Thus, quantification of drug compounds using chromatographic separation techniques is quite challenging. For this reason, current clinical quantification of drugs of abuse is carried out by hydrolysis of the phase two metabolites and some phase one metabolites (either by acid or enzymatic hydrolysis) in order to convert the metabolite back to the parent compound and subsequently, LCMS measurement of only the parent compound is necessary. The total concentration of the metabolites and parent drug are reported together as a single value because the metabolites have been converted back to the parent during hydrolysis. Either acid hydrolysis or enzymatic hydrolysis requires a treatment step followed by up to 2 hours of incubation at an elevated temperature. Thus, the hydrolysis procedure adds extra time and cost to each analysis.
“In accordance with the above discussion, there is a need in the art for a quick and reliable chromatographic separation and analysis methods–such as an LCMS method that does not require an additional hydrolysis step–for routine clinical measurements of drug compounds. There is also a need for new chromatographic designs that are able to implement the new methods. Unfortunately, the conventional instrumentation required for LCMS is technically complex and not well suited to the typical hospital clinical lab or medical lab technician. These clinical labs have not adopted LCMS diagnostics and, instead, generally use alternative diagnostic techniques, including automated immunoassay. Alternatively, the clinical labs may send the samples out to a central reference laboratory for analysis.
“Recently, however, an automated analyzer that is suitable for routine clinical and hospital use has recently been described in international patent application (PCT) publication WO 2012/058632 A1 titled ‘Automated System for Sample Preparation and Analysis’ which is hereby incorporated by reference herein in its entirety. Adaptation of such an automated analyzer so as to further include routine analyses for drugs of abuse using one of the conventional hydrolysis methods would require either off-line hydrolysis followed by transfer of the treated samples to the automated analyzer or else on-board automated robotic hydrolysis. Implementation of either of these modifications would require additional hardware and increased overall system complexity. Further, a recent study (Wang et al., ‘Incomplete Recovery of Prescription Opioids in Urine using Enzymatic Hydrolysis of Glucuronide Metabolites’, Journal of Analytical Toxicology, Vol, 30, Oct. 2006, pp. 570-575) has concluded that, with regard to at least opioids in urine samples, acid hydrolysis liberates a greater proportion of the parent drug compounds and introduces less variability than enzymatic hydrolysis. To implement the evidently preferred method of acid hydrolysis for such samples would require additional expensive safeguards to prevent lab personnel from being exposed to hazardous reagents. Therefore, there is a special need in the art for a quick and reliable chromatographic separation and analysis method–such as an LCMS method that does not require an additional hydrolysis step–that may be employed using an automated clinical analyzer to make routine clinical measurements of drug compounds,”
As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors’ summary information for this patent application: “In order to address the above-noted needs in the art, the inventors provide methods and apparatus which permit removal of hydrolysis steps for quantifying drug compounds in biological samples. Methods in accordance with the present teachings include measuring metabolites and parent compounds using two columns in a single LCMS run and then reporting the sum of the all the related compounds so as to calculate concentrations of the original parent compounds and derive the same values as would be generated by performing traditional hydrolysis.
“Accordingly, in a first aspect of the invention, a liquid chromatography apparatus for detecting and quantifying a plurality of analytes in a sample is provided, wherein the apparatus comprises: a first chromatographic column fluidically coupled to a source of the sample and to a source of a first chromatographic mobile phase solvent; a second chromatographic column fluidically coupled to the first chromatographic column; a source of a second mobile phase solvent fluidically coupled between the first and second chromatographic columns; and a detector fluidically coupled to the second chromatographic column, wherein the first chromatographic column is configurable to receive, in an analyte trapping step, the first mobile phase solvent and the sample and to retain a first portion of the plurality of analytes therein and to pass a second portion of the plurality of analytes therethrough, wherein the second chromatographic column is configurable, in the analyte trapping step, to receive the second portion of the plurality of analytes and the first and second mobile phase solvents and to retain the second portion of the plurality of analytes therein, and wherein the detector is arranged to receive the second and first portions of the plurality of analytes in a first and in a second elution step, respectively.
“In a second aspect of the invention, a method for analyzing and quantifying a panel of drugs in a clinical sample is provided, wherein the method comprises: trapping a first portion of drug parent compounds and their metabolites on a first chromatographic column; trapping a second portion of the drug parent compounds and their metabolites on a second chromatographic column; separately eluting the first and second portions of the drug parent compounds and their metabolites from the first and second chromatographic columns; detecting concentrations of each of the drug parent compounds and metabolites eluted from each of the first and second chromatographic columns with a detector; and summing the detected concentration of each drug parent compound together with the detected concentrations of all of its respective analytes so as to derive, a respective total concentration of each drug in the sample.
“In another aspect of the invention, there is provided an automated sample preparation and analysis system comprising: (a) an automated sample preparation station for preparing a plurality of samples in accordance with a plurality of assays that are selected from a database containing a plurality of unique assays; (b) a sample analysis station for automatically performing the plurality of assays; and © a transport mechanism for transporting prepared samples from the sample preparation station to the sample analysis station, wherein the sample analysis station comprises: (i) a source of a first chromatographic mobile phase solvent; (ii) a source of a second chromatographic mobile phase solvent; (iii) a first chromatographic column fluidically coupled to the first chromatographic mobile phase solvent and configured to receive a portion of the prepared samples from the sample transport mechanism, (iv) a second chromatographic column fluidically coupled to the first chromatographic column and to the source of the second chromatographic mobile phase solvent; and (v) a detector fluidically coupled to the second chromatographic column, wherein the first chromatographic column and the second chromatographic column are operable to trap, respectively, a first portion and a second portion of a panel of drug compounds and their metabolites during a trapping step and to elute, respectively, the first and second portions of the drug compounds and their metabolites to the detector.
BRIEF DESCRIPTION OF THE DRAWINGS
“The above noted and various other aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings, not drawn to scale, in which:
“FIGS. 1A-1D are diagrams of a first chromatographic system in accordance with the present teachings, illustrating sampling and analyte trapping and elution steps;
“FIGS. 1E-1G are diagrams of a second chromatographic system in accordance with the present teachings, illustrating analyte trapping and elution steps;
“FIG. 2 is a schematic depiction of a multiport valve as may be employed in the chromatographic system depicted in FIGS.
“FIG. 3A-3D are diagrams of a third chromatographic system in accordance with the present teachings, illustrating sampling and analyte trapping and elution steps;
“FIG. 4 is a diagram of a fourth chromatographic system in accordance with the present teachings;
“FIG. 5A is a perspective view of an automated sample preparation and analysis system in accordance with some embodiments in accordance with the present teachings:
“FIG. 5B is a perspective view of another automated sample preparation and analysis system in accordance with various other embodiments in accordance with the present teachings;
“FIG. 6 a general diagrammatic view of the components automated sample preparation and analysis system;
“FIG. 7A is a top view of the automated sample preparation and analysis system of FIG. 5A;
“FIG. 7B is a top view of the automated sample preparation and analysis system of FIG. 5B;
“FIG. 7C is a perspective view of the components of the automated sample preparation and analysis system of FIG. 5A;
“FIG. 8 is a schematic view of a sample preparation station and a transport assembly of the automated sample preparation and analysis system of FIG. 5A;
“FIG. 9A is a schematic view of a first system comprising a sample preparation station and a sample analysis station of an automated sample preparation, and analysis system in accordance with one embodiment of the present teachings;
“FIG. 9B is a schematic view of a second system comprising a sample preparation station and a sample analysis station of an automated sample preparation and analysis system in accordance with another embodiment of the present teachings;
“FIG. 10 is a flowchart, of a general method for operating a chromatographic system in accordance with the present teachings;
“FIG. 11 is a schematic diagram of a modular cartridge configuration which may be used for insertion and removal of pairs of analytical columns matched in accordance with the present teachings; and
“FIG. 12 is a set of chromatograms of various analytes in urine samples, as observed using the system illustrated in FIG. 1.”
For additional information on this patent application, see: HERMAN, Joseph; FAIR, Sarah J.; ARGOTI, Dayana. Methods and Apparatus for Analysis of Phase-I and Phase-II Metabolites and Parent Compounds without Hydrolysis. Filed December 21, 2012 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=\%2Fnetahtml\%2FPTO\%2Fsearch-adv.html&r=3059&p=62&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626
Keywords for this news article include: Patents, Hospital.
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