WO2008027772A2

WO2008027772A2 - Ultrafast immunoextraction/displacement assays

Info

Publication number: WO2008027772A2
Application number: PCT/US2007/076541
Authority: WO
Inventors: David S. Hage; Corey M. Ohnmacht
Original assignee: Board Of Regents Of The University Of Nebraska
Priority date: 2006-08-30
Filing date: 2007-08-22
Publication date: 2008-03-06
Also published as: WO2008027772A3

Abstract

An analytical method for determining the concentration of an analyte or free analyte fraction in a sample that uses affinity chromatography in conjunction with labeled analyte analogs. In the method a sample containing an analyte is applied to an affinity column capable of selectively extracting the analyte in the millisecond time domain. The concentration of free analyte is then determined by a chromatographic affinity assay that correlates the analyte concentration to the quantity of labeled analyte competitively displaced from the column. The method of the invention is useful for determining concentrations of an analyte or a free analyte fraction in a single sample or in multiple samples on a short time-scale. Automation of such a method is further contemplated.

Description

Analysis of Free Drug Fractions Using Near Infrared Fluorescent Labels and UItrafast Immunoextraction/Displacement Assays

Cross-Reference to Related Applications The present application claims the benefit of U.S. Provisional Application Serial No. 60/823,964, filed August 30, 2006, and entitled "Analysis of Free Drug Fractions Using Near Infrared Fluorescent Labels and UItrafast

Immunoextraction/Displacement Assays", which is incorporated herein by reference in its entirety to the extent that it is not contrary to the teachings herein. Statement Regarding Federally Sponsored Research or Development

This invention was made with government support under GM044931 awarded by the National Institute of Health. The government has certain rights in the invention. Appendix Not Applicable Background of the Invention

Field of the Invention

The invention relates generally to analytical methods for determining the concentration of an analyte or free analyte fraction in a sample. The invention particularly relates to determining the concentration of an analyte or free analyte fraction in a sample, comprising a free and bound analyte fraction, by a quantitative immunoassay comprising affinity chromatography for extraction of the free analyte fraction on the millisecond time scale and the use of labeled analyte analogs for detection of the quantity of free analyte extracted. The methods of the invention are useful for determining concentrations of an analyte or a free analyte fraction on a short time-scale in a single sample or in multiple samples.

Related Art

Many drugs, hormones, and toxins exist in two forms when present in the bloodstream: 1) a fraction that is reversibly or non-covalently bound to serum proteins or other agents in blood and 2) a fraction that is non-bound, or free, in solution.¹'² Because the two states are reversible, the solutes in one fraction are continually exchanging with those in the other. In biological systems this process is constant and proceeds to form a dynamic equilibrium between the free and bound fractions. It is generally hypothesized that the free fraction of such substances represents the biologically-active form. Substances, such as drugs, are understood to be accessible in the free form for interacting with their primary molecular target and achieving their therapeutic effect. For example, being in the free form enables pharmaceutical compounds to cross cell membranes or interact with cell receptors and other target ligands.³ Although substances bound to serum components can importantly provide a mechanism for drug delivery through the bloodstream,⁴ the fraction bound to serum components is not regarded as available for interacting with the primary molecular target. Therefore analysis of free fractions of these substances in the presence of serum components is of particular interest in clinical chemistry and pharmaceutical science for therapeutic drug monitoring, pharmacological studies, and measurements of drug-protein binding.¹'²'⁵-⁶'¹³-¹⁵'²⁰'²¹'²⁷'⁵⁶'⁵⁷

For many substances it is possible to use the total concentration of the substance in blood or serum to estimate the concentration of the free fraction by assuming that a constant relationship exists between these two values. However certain changes in physiology may drastically alter the relationship between these two values and this method will not accurately reflect the concentration of the free fraction, nor suggest the availability of the biologically-active form. For example, factors such as surgery, trauma, malnutrition, pregnancy, illness (e.g., cancer, renal failure or liver disease) and age (e.g., in newborns or the elderly) can cause large fluctuations in the concentration of binding proteins present in blood. The extent of a drug's binding with serum proteins can vary as a result.²' ⁹ A large fluctuation in the quantity of binding proteins can shift the equilibrium between the free and bound forms and thereby change the concentration of the free fraction, even as the total concentration of the drug remains constant. These factors can lead to individual variations in free drug fractions.⁵ Similar shifts in drug-protein binding can occur in situations where several drugs and/or endogenous agents compete for the same binding proteins and make estimates of the free fraction based on total concentration inaccurate. Situations in which a drug is in excess of its binding proteins also presents problems for estimating the free fraction based on total protein concentration. When a drug has a high total concentration relative to the concentration of its binding proteins, a nonlinear relationship between the drug's total and free levels may result and confound estimates of the free fraction based upon total protein concentrations. These types of situations can make it difficult to correlate the total concentration of a drug with its free fraction, creating a need for an accurate and precise means of routinely measuring free drug fractions to help identify and correct for such variations.

Direct analytical methods have been developed for the measurement of a drug's free fraction in blood, plasma, or serum ^{• ■ • "} Examples include techniques based on equilibrium dialysis, ultrafiltration, restricted access media (RAM) HPLC columns, and the use of natural filtrates.⁶'¹⁰'¹⁴'¹⁵'¹⁸'¹⁹'²¹ Although not relying on assumptions of total concentration, these methods suffer from inherent inaccuracies, long analysis times from lengthy procedures, or protocols requiring much manual manipulation. A major problem with these techniques is that the analysis often involves the use of an additional binding reagent or separation process that undesirably and non-specifϊcally interacts with the free or bound fraction (e.g., binding of drugs to dialysis or filtration membranes)¹⁹ thereby altering the equilibrium. Other techniques (e.g., methods based on restricted access media) have been used with only drug/protein mixtures or give only an incomplete separation of the free and bound forms of this drug, making it difficult to obtain accurate free fraction measurements or to use such methods with real clinical samples.¹'²'¹⁶'²⁷ Many of these techniques are also limited to certain types of analytes, as is the case with natural filtrates, such as tears or saliva. Additionally, techniques with long analysis times results in measurement bias. Even on the order of several seconds, sufficient time elapses to allow the release of a significant amount of solute from the bound fraction that is then detected along with the original free fraction.¹⁹ Consequently, an error in the determination of the concentration of the original free fraction results. Lastly, many of these analytical methods require manual manipulation of the sample for signal detection after the assay has been performed.

The detection of signal from any assay poses further challenges in cases when the solute cannot be measured directly because it has no unique properties available for detection that distinguish it from interfering substances in the background. Even when a unique property for detecting the signal is available, it may require further manipulation in the form of a post-assay or secondary reaction. A post-assay reaction may require additional manipulation if it cannot be simultaneously performed under the primary assay conditions or if it cannot by chemically coupled or mechanically linked to the primary assay. Furthermore post-assay reactions may also require additional effort in performing controls to establish the baseline of the background or preparing the sample for the post-assay reaction. Lengthy post-assay procedures are undesirable in situations where a rapid determination of concentration needs to be made or when the concentrations of many samples need to be determined. Lengthy post-assay procedures may also be less accurate in cases where the analyte is unstable.

Given the state of the field, a need exists for a method for determining the free fraction of a substance without perturbing the equilibrium between the free and bound fractions of the substance. Additionally, a need exists for a method that is highly specific for detection and can be employed to determine the free fraction of a vast number of different substances. It is desirable that the method utilize a fast reaction time to minimize changes in equilibrium. In addition, it is desirable that signal detection in the method be sensitive and accurate over the expected range of concentrations requiring determination, be compatible with biological samples, provide rapid detection, and minimize the amount of manual manipulation.

Summary of the Invention

The invention relates generally to analytical methods for determining the concentration of a free analyte fraction in at least one sample, the sample comprising a bound analyte fraction and the free analyte fraction, the free analyte fraction and the bound analyte fraction comprising free analyte and bound analyte, respectively. More particularly the invention relates to analytical methods for determining the concentration of an analyte or a free analyte fraction in a sample using affinity chromatography (such as performed using immobilized antibodies) in conjunction with labeled analyte analogs. In the method of the invention, an affinity column is prepared by applying a labeled analyte, the labeled analyte being a labeled analog of the free analyte, in or approaching a saturating quantity to an affinity column having an active layer that binds at least some of the labeled analyte and selectively binds the free analyte relative to the bound analyte, wherein the active layer separates the free analyte fraction from the bound analyte fraction of the sample in the millisecond time domain. The excess labeled analyte, if any, is removed from the affinity column having the active layer with the bound labeled analyte. The sample is applied to the affinity column, having the active layer with the bound labeled analyte and from which excess labeled analyte has been removed, thereby producing a displacement of the labeled analyte from the affinity column. A signal caused by the displacement of the labeled analyte from the affinity column by binding of the free analyte fraction of the sample to the affinity column is detected, and the concentration of free analyte present in the sample is determined from the signal.

The invention also relates generally to analytical methods for determining the concentration of a free analyte fraction in at least two samples, each sample comprising a bound analyte fraction and the free analyte fraction, the free analyte fraction and the bound analyte fraction comprising free analyte and bound analyte, respectively. More particularly the invention relates to analytical methods for determining the concentration of an analyte or a free analyte fraction in a sample using affinity chromatography (such as performed using immobilized antibodies) in conjunction with labeled analyte analogs. In the method of the invention, an affinity column is prepared by applying a labeled analyte, the labeled analyte being a labeled analog of the free analyte, in or approaching a saturating quantity to an affinity column having an active layer that binds at least some of the labeled analyte and selectively binds the free analyte relative to the bound analyte, wherein the active layer separates the free analyte fraction from the bound analyte fraction of the sample in the millisecond time domain. The excess labeled analyte, if any, is removed from the affinity column having the active layer with the bound labeled analyte. The samples are applied in sequence to the affinity column, having the active layer with the bound labeled analyte and from which excess labeled analyte has been removed, thereby producing a displacement of the labeled analyte from the affinity column. The signals caused by the displacement of the labeled analyte from the affinity column by binding of the free analyte fraction of the sample to the affinity column are detected in the sequence of their application, and the concentration of free analyte present in each sample is determined from the signals corresponding to the sequence in which the samples were applied.

It is in view of the above problems that the present invention was developed. The speed with which the antibody-based affinity reaction of the method works addresses problems related to loss of accuracy from long reaction times that perturb the equilibrium of the free and bound fractions in a sample. In addition, use of a labeled analyte analog which couples a detection method to the antibody-based affinity reaction provides several advantages. Signal detection can be rapidly obtained after the primary reaction when the labeled analyte analog is a NIR fluorescent dye because a detector can be placed downstream when a column is used. The use of NIR fluorescent dyes is compatible with biological fluids because biological fluids have minimal background contaminants that interfere in the near infrared spectrum. Moreover, the sensitivity and linear response over biologically relevant concentrations is desirable with detection limits in the femtomole range.²⁹ Lastly, use of a detector minimizes any post-reaction manipulation.

A person of skill in the art can adapt this method to determine the concentration of free analyte fractions for a number of substances by use of appropriate reagents and optimization of the conditions under which the reagents are used.

Brief Description of the Drawings

The accompanying drawings, which are incorporated in and form a part of the specification, illustrate the embodiments of the present invention and together with the description, serve to explain the principles of the invention. In the drawings:

Figure 1 illustrates a drawing of a typical microcolumn that may be employed in the method of the invention.

Figure 2 illustrates change in column void time with column length and solvent flow rate for 2 mm ID HPLC columns packed with porous silica. These results assume an overall porosity of 0.80 within the column (i.e., 80% of the column volume is occupied by the mobile phase). Using a column with an inner diameter of 1 mm or 4 mm gives similar results but with the vertical position of the lines in this graph being lowered or raised by 4-fold, respectively.

Figure 3 illustrates the reproducibility of stationary phase content in a sandwich microcolumn as a function of the number of injections which were used to apply a fixed amount of an immobilized hemoglobin support to a 2.1 mm ID x 620 μm microcolumn. These results represent the average of triplicate analyses.

Figure 4 illustrates a general scheme for the method of the invention ultrafast immunoextraction/displacement assay (UFIDA). The illustration in Figure 4 A represents the steps and the illustration in Figure 4B depicts a typical chromatogram for such an assay. The example in Figure 4B is based on the phenytoin system described in Example 9.

Figure 5 illustrates a scheme for the synthesis of a phenytoin conjugate containing a label based on a near-IR fluorescent dye. The illustration in Figure 5A shows the reaction for the preparation of 3-N-amino-5,5-diphenylhydantoin (ADPH), while the second illustration in Figure 5B shows the reaction used to conjugate NHS-activated IRDye 78 (LI-COR Biosciences) to ADPH to yield the final labeled phenytoin-dye conjugate. The dye shown in this example is similar to the proprietary dye used in one embodiment of the invention.²⁵

Figure 6A illustrates the emission spectrum for serum and labeled phenytoin in the presence of serum or pH 7.4, 0.067 M phosphate buffer and Figure 6B illustrates chromatograms for a UFIDA method performed for samples containing free phenytoin concentration of 4 to 8 μM injected at 6 min after the application of labeled phenytoin. The chromatogram shown in Figure 6B was obtained under the same conditions for a 5 μL, 550 μM sample of HSA₅ as monitored using UV/absorbance detection at 205 run. The emission spectra in Figure 6A were obtained using a 100 μM solution of labeled phenytoin in serum or buffer at an excitation wavelength of 770 nm.

Figure 7A illustrates the effect of injection time on the application of phenytoin samples to the UFIDA system and Figure 7B illustrates the use of sequential sample injections in this assay. The experiments with the injection time were performed using a 5μL, 4 μM sample injected at 2, 4, 6 and 12 min after application of the phenytoin-dye conjugate. The sequential injection studies were performed using a sample containing 30 μM phenytoin and 550 μM HSA injected at 6, 9, 12 and 14 min after application of the phenytoin-dye conjugate. Other experimental conditions are given in Example 10.

Figure 8 illustrates a calibration curve based on displacement peak area for phenytoin in the UFIDA method. The experimental conditions are given in Example 9. The best-fit line over the linear range in was y = 5.58 (± 0.02) x + 0.010 (± 0.106), with a correlation coefficient of 0.9999 (n = 10).

Detailed Description of the Preferred Embodiments

Abbreviations and Definitions:

"Analyte'Or "Target Analyte" are used interchangeably herein, and shall mean the component of the sample that binds to the binding agent present in the active layer of the column. The analyte will typically comprise the free fraction of a drug, hormone, toxin, metal ion, fatty acid, bilirubin or any other endogenous or exogenous compound. Additionally, the analyte may also be any other inorganic or organic compound capable of being separated from the sample, as described herein.

"Binding Agent", as utilized herein, shall mean the agent in the active layer capable of selectively binding the target analyte. "Binding Compound", as utilized herein, shall mean the compound that the bound fraction binds in a sample or solution. Typically, the binding compound comprises a protein, cell or any other endogenous or exogenous compound.

"Bound Fraction" or "Bound Analyte Fraction" are used interchangeably herein, and shall mean the portion of the analyte which is bound to a binding compound.

"Free Fraction" or "Free Analyte Fraction" are used interchangeably herein, and shall mean the portion of the analyte which is not bound to a binding compound.

"Immunoaffinity Column" or "Immunoextraction Column" are used interchangeably and refer to a chromatographic column that contains antibodies or antibody-related binding agents.

"Labeled Analyte", "Labeled analyte analog", "Labeled analog" are used interchangeably herein, and shall mean an analog of the free analyte comprising the free analyte irreversibly or covalently coupled to a label detectable by standard analytical methods.

"Millisecond Time Domain", as utilized herein, shall mean any amount of time less than one second.

"Sandwich column", "sandwich microcolumn", or "microcolumn" are used interchangeably herein, and shall mean an embodiment of the invention wherein the column contains a top inert layer, a bottom inert layer and an active layer between the two inert layers.

"Sample" or "Liquid" are used interchangeably herein and shall mean the mixture applied to the column containing the analyte. In addition to the analyte, the sample (liquid) generally also contains a loading buffer. Any loading buffer may be employed to the extent that the buffer does not interfere with the separation process. The sample may comprise any mixture with an analyte. Typically, however, the sample will be comprised of a biological fluid such as blood, plasma, urine, cerebrospinal fluid, tissue samples, or intracellular fluid.

"Uniform Manner", as utilized herein, shall mean loading the layers of the column in a manner such that these layers have a substantially equal distribution of support in both a horizontal and vertical direction.

"Ultrafast immunoextraction/displacement assay", as utilized herein, shall mean a method for determining the concentration of analyte in a sample in which a small immunoextraction column is used to bind a measurable amount of the free analyte (e.g., phenytoin) on a time scale that is sufficiently brief to avoid any appreciable dissociation of the analyte from its binding compounds in the sample (e.g., HSA). At the beginning of this method, an excess of a labeled analog of the analyte is applied to the small immunoextraction column. Some of this labeled analog will bind to antibodies in the column while the remainder will be washed away prior to sample injection. When the sample is later injected, the free fraction of the analyte in this sample will have the opportunity to compete with any labeled analog that is momentarily dissociated from immobilized antibodies in the column. The result is a displacement peak for the labeled analog, which gives a signal proportional to the drug's free fraction.

BSA=Bovine Serum Albumin

EI-MS=Electron Ionization Mass Spectrometry

FPLC=Fast-Protein Liquid Chromatography HPLC=High-Performance Liquid Chromatography

HSA=Human Serum Albumin

NIR=Near-infrared

RAM=Restricted access media

UFIDA= Ultrafast immunoextraction/displacement assay

In view of the foregoing, it will be seen that the several advantages of the invention are achieved and attained. Many compounds, as stated above, exist in two distinct forms as they pass through the bloodstream: 1) a fraction that is reversibly or non-covalently bound to proteins or other binding compounds in serum or related samples and 2) a fraction that is non- bound, or free, in the sample. The free and bound fractions are present in a dynamic state, in which solutes in one state are continually exchanging with those in the other. Applicants have discovered a method to determine accurately the free analyte fraction of a sample comprising a free analyte fraction and a bound analyte fraction. The method of the present invention employs the use of affinity columns to extract the free analyte fraction from the sample in the millisecond time domain and the use of labeled analyte analogs to detect the quantity of free analyte bound, which correlates directly with the concentration of the free analyte fraction. The capability of extracting the free fraction in this time domain allows the free fraction to be separated without perturbing the equilibrium between the free and bound fractions. Applicants' discovery, accordingly, solves a major problem associated with the long separation times that compromise current detection techniques. The use of labeled analyte analog which can be easily detected without sacrificing accuracy also represents an improvement. This detection method solves problems associated with other methods of detection, including incompatibility with biological samples and the requirement of an additional post-assay method for determination. The detection method is relatively rapid and minimizes manual manipulation. Minimizing manual manipulation contemplates automation of the method for increased through-put processing of samples. The concentration of the free fraction is determined from the signal detected employing standard analytical methods.

1. Affinity Column Design and Construction

The method of the present invention employs the use of a column to separate the free fraction of an analyte from the sample in the millisecond time domain. Figure 1 depicts a typical column that may be employed in the method of the invention. As shown in Figure 1, the column 1 generally has a tubular configuration with a first end

2, a second end 3, a passageway 4 therebetween, and retaining means 5 and 6 at the first and second ends, respectively, of the column 1. However, the column 1 may comprise any number of different shapes, all of which are embodiments of the present invention. The retaining means 5,6 typically comprises mesh or small-pore material that acts to hold the support particles or support material within the column while allowing fluid flow therethrough. The column 1 may also contain end fittings 7 and 8 at the first and second ends, respectively, of the column 1 used to connect the column to the chromatographic system. The column 1 comprises a thin active layer 9 to facilitate separation of the analyte from the sample in the millisecond time scale and typically a single inert layer in one embodiment, to several inert layers in additional embodiments. Figure 1 illustrates an embodiment with a top inert layer 10 and a bottom inert layer 11.

A salient feature of the current invention is the capability of removing a significant amount of the free fraction of a particular analyte from a sample without release of the analyte from its protein-bound fraction. In order to accomplish this task, as delineated above, the extraction process is preferably accomplished in the millisecond time domain. The column design described herein is particularly suitable for this application because, due in part to its relatively thin active layer, it can extract the free fraction within this time range. As used herein, "length" or "thickness" of a layer refers to the dimension of the layer generally parallel to the direction of flow. As illustrated by Figure 2 (plot derived by calculation), only columns with active layer lengths in the range of about 100 microns to about 1 millimeter allow separation in the millisecond time domain when employing standard HPLC flow rates of about 0.1 to about 1.0 mL/min. Accordingly, to achieve separation in this time range, the preferred columns employed generally comprise an active layer that may be less than about 100 microns in thickness. Typically, however, the active layer is from about 10 microns to about 1.1 millimeters in thickness and preferably, is at least approximately 60 microns in thickness. Applicants have found that active layers with these dimensions, depending upon the specific sample applied, are generally capable of extracting an analyte in about 1 to about 500 milliseconds. Columns having active layers capable of an analyte in less than about 200 milliseconds are preferred. In addition to rapid extraction of the free fraction, an additional salient feature is the ability of the active layer to bind the target analyte with both a high degree of selectivity and with a relatively high binding affinity. The active layer, therefore, typically comprises support particles derivatized with any binding agent possessing selectivity and having a high binding affinity for the target analyte. Generally, any protein or compound with high affinity for the analyte may be used as the binding agent. Preferably, such binding affinity is from about 10² to about 10⁶ M^"1 or greater. In a preferred embodiment, the binding agents are antibodies raised against the target analyte. The antibodies can be either monoclonal or polyclonal. However, monoclonal antibodies are generally employed in applications where a higher degree of selectivity is desired and polyclonal antibodies are more typically utilized in applications where a higher degree of binding affinity is desired. Examples of other suitable binding agents include nucleic acid ligands (e.g. aptamers), synthetic molecular imprints, antibody fragments (e.g. Fab fragments), antibody related molecules (e.g. chimeras or F_v chain fragments), and recombinant proteins that act as antibody mimics. The binding agent, once selected, may be isolated in accordance with any generally known method.

The binding agent can be derivatized to the support particles or support material by any method generally known in the art. However, the method preferably immobilizes the binding agent to the support particles or support material in a manner such that a relatively high percent of the binding agent is active (i.e., binds the target analyte) after the immobilization process. Suitable immobilization methods for protein ligands, for example, include the Schiff base method and the carbonyldiimidazole method. The Schiff base method is generally employed when immobilizing the binding agent through free amine groups. However, when the binding agent comprises antibodies, applicants have found that a more preferred approach is immobilization through the antibodies' carbohydrate region because this generally results in an active layer with a higher number of active binding sites compared to when immobilization is performed through free amine groups. Any method known in the art for immobilization via carbohydrate regions may be employed. The overall binding capacity of the column is also significant because it affects both the time and efficiency of extraction by the active layer. The binding capacity of the column, in part, is determined by the number of active binding sites present in the active layer. Preferably, the minimum number of active binding sites in the column is such that the ratio of active binding sites in moles to the amount in moles of free analyte present in sample is at least about 1 :1, and even more preferably, at least about 10:1. However, still more preferably, support particles or support material in the active layer will be derivatized with the maximum concentration of active binding agent achievable so that the column has the largest binding capacity practical.

The active layer, additionally, may comprise a number of different support particles or support material. The support particles or support material, as detailed above, function primarily as a surface or a structure to immobilize the binding agent. When using support particles, the diameter of the particle, however, is significant and should be considered because it affects both the length of the active layer and the amount of binding agent that may be immobilized in the active layer (i.e., binding capacity of the column). Preferably, the diameter of the particle size is smaller than the length of the desired active layer. Applicants have found that a preferred particle diameter is 7 μm or less than about 10 times to about 20 times the length of the active layer. Particles within this size range facilitate uniform packing of the layer by allowing small packing defects to average out and produce a more uniform packing cross-section for the support. In addition, the support particles should be able to tolerate the flow rates and pressures needed in order to obtain the desired sample contact time with the active layer. The properties that affect the pressure and flow rate that may be tolerated by the support particles include the diameter of the particle, the particle's shape and the porosity of the particles. Suitable support particles include porous or nonporous glass, silica and other inorganic supports (e.g., alumina or zirconia), carbohydrate-based supports (e.g., beaded agarose), and polymeric supports (e.g., polymethacryltate or polystyrene based resins); however, one generally skilled in the art of chromatography can select other appropriate support particles or support materials, including monolithic supports. The column may comprise a single inert layer or several inert layers, depending upon the application. However, common features shared by all inert layers, irrespective of their number or position within the column, is that they typically should have no substantial interaction with the target analyte, and should preferably be mechanically stable under the flow rate and pressures employed during the separation process.

Preferable materials for construction of the inert layer include diol-bonded silica, diol- bonded glass beads, agarose beads, hydroxylated perfusion media, and glycol coated perfusion media. The various inert layers may be constructed from the same support particles and support materials or different support particles and support materials. However, it is usually preferred for the sake of convenience in loading the column that the inert layers comprise the same support particles or support materials.

The layers in the column may comprise either an active layer alone, or an active layer and a single inert layer on top of the active layer (wherein the active layer is in communication with the second end retaining means) such that liquid first passes through the inert layer and then passes through the active layer. The utilization of a single inert layer in this manner is especially suitable for applications where the liquid (containing the sample) is to be applied in only a single direction to the active layer and the column. The inert layer in this application preferably occupies the entire length of the column between the beginning of the first end of the column to the beginning of the active layer so that the entire column is filled with support particles or support material. Applicants have found that having the entire column filled with support particles or support material increases both the speed and efficiency of separation. Additionally, the inert layer in this application also preferably acts to distribute the injected sample evenly across the diameter of the column before the sample reaches the active layer. This allows for a more uniform application of the sample to the relatively thin active layer.

The layers in the column may also comprise an active layer sandwiched between a top and a bottom inert layer. As utilized herein, the term "top inert layer" shall mean the layer that liquid first passes through prior to reaching the active layer and "bottom inert layer" shall mean the layer where liquid passes after it exits the active layer. The column preferably comprises both a top and bottom inert layer for applications where liquid is to be applied in two directions to the active layer and the column. At any given time, the flow of liquid through the column is generally only in a single direction. However, it is sometimes preferable to alternate the flow of liquid through the column in order to help wash out any impurities that may have built up at the top of the column during the application of liquid. The top inert layer in this application serves the same role as discussed above for the application employing a single inert layer e.g. more efficient separation. However, applicants have found that it is preferable to include the bottom inert layer, even in applications where fluid flow is in only a single direction, because its inclusion increases the useful life of the active layer by preventing loss of support particles or support material.

Any thickness of the inert layers may be used and thickness of the inert layers does not affect the time needed to separate the free fraction of an analyte from the sample. In general, as stated above, the top inert layer is preferably the length that remains between the beginning of the column and the beginning of the active layer. And, the bottom inert layer, if it is present, is generally from about 1 to about 5 times the length of the active layer. Typically, the top inert layer is thicker than the bottom inert layer.

Any type of housing for the column may be used, although the column housing preferably employs components made of materials that are substantially inert to biological fluids and in particular, substantially inert to the analyte so as not to interfere with the separation process. Accordingly, any material that is substantially inert may be employed to construct the column. Suitable materials include stainless steel, polypropylene, certain plastics and fused silica.

Although any dimensions of the column may be used, any size of column may be utilized to the extent that the total column length is preferably greater than the length of the active layer. The total column length and diameter also preferably allow the use of sufficiently fast flow rates and pressures to achieve the desired contact time between the sample and the active layer. Preferably, the column has an internal diameter of about 50 microns to about 2 centimeters and a length of about 0.2 millimeters to about 2 centimeters. In a particularly preferred embodiment, the column has an internal diameter of about 0.5 to about 2.1 millimeters and a length of about 1 millimeter to about 2 centimeters.

When using support particles, applicants have found that thin active layers may preferentially be obtained by loading the support particles comprising the layer into the column via a plurality of injections, as described in detail below (e.g., see Figure 3). The normal method of loading a column, applying the support particles in one application to the column with the amount of support particles being in excess of that which is needed to fill the column, is sufficient for standard size chromatography columns because due to their size, reliable packing of the support particles may be achieved. Applicants, however, have found that loading the support particles in a single injection, for columns with dimensions described herein, generally does not result in an active layer capable of consistently achieving separation of an analyte from a sample in the millisecond time scale.

Accordingly, the layers are preferably loaded into the column by a plurality of injections of slurry comprising the support particles. The slurry may be injected into the column employing any apparatus generally known for injecting a slurry into a column, for example, a closed-loop sample application system with either a manual injection valve or an automatic injection system may be utilized. The slurry, in addition to support particles, also preferably comprises a packing solvent or buffer. Any packing solvent may be employed to load the slurry into the column; however, the solvent preferably will not harm the binding agent present in the active layer. One skilled in the art of chromatography can readily select both an appropriate apparatus to inject the slurry and appropriate packing solvents.

Applicants have also found, as illustrated by Figure 3, employing a larger number of injections and less support per injection achieves a more controlled delivery of support particles because statistical variations that occur during the delivery of small amounts of support particles to the column are averaged out. This is particularly true as layer thickness decreases. Uniform packing of support particles in the layers, especially the active layer, is preferable because it provides more reproducible results for injected samples by allowing parts of the sample that are injected at different locations along the diameter of the column to achieve consistent sample contact times with the active layer. Accordingly, the number of injections to introduce a layer into the column is generally from about 10 to about 100. More preferably, the number of injections to introduce a layer is from about 30 to about 40 when the layer length is from about 100 to about 500 microns, and is from about 60 to about 80 injections when the layer length is from about 60 to about 100 microns in length.

The slurry density (milligrams of support particles per milliliter of packing solvent), or amount of support particle applied to the column per injection, will vary greatly depending upon the desired thickness of the layer. Typically, however, the slurry density will be from about 0.1 to about 20 milligrams of support particles per milliliter of packing solvent and more preferably, will be from about 1 to about 5 milligrams of support particles per milliliter of packing solvent. In general, the inert layer(s) and active layer are loaded at approximately the same slurry density. One of ordinary skill in the art can readily determine the appropriate slurry density needed to achieve a layer having a particular thickness when employing a specific number of injections.

The desired slurry density, once selected, is preferably maintained throughout column injection in order to facilitate uniform layer packing. To maintain consistent slurry density, the slurry typically undergoes shaking between injections to ensure that the support particles are uniformly distributed in the slurry. It is also preferable to monitor the turbidity of the slurry at a wavelength of approximately 800 nm to ensure the amount of support particles per milliliter remains constant. Furthermore, typically the slurry density is calculated at numerous points during injection by comparison to slurries of known density employing the same support particles.

Applicants have also found, in addition to loading the support particles by a plurality of injections, that varying the flow rate and pressure during column loading also serves to provide a more uniform and thin active layer. In a particularly preferred application, the pressure and flow rate are increased for a short period of time near the beginning and end of the loading of each layer (as illustrated in Table 1). This increased pressure and flow rate facilitates compression of the layer and distributes the support particles within the layer evenly across the diameter of the column. In a typical column loading procedure, for example, the flow rate of slurry injection into the column is between about 3 mL/min and about 5 niL/min, with the higher flow rate occurring generally at the beginning and end of the loading of each layer. Additionally, pressure during column loading is typically maintained between about 2000 and about 4000 psi, with a higher pressure preferably occurring at the beginning and end of the loading of each layer. Any flow rates and pressures may be utilized to load each layer of the column and accordingly, may be varied significantly from the general examples provided herein depending upon the particular application.

A general procedure for loading a 1.0 cm immunoaffinity column comprising an active layer between a top and bottom inert layer is provided. The procedure is for illustrative purposes only and shall not be construed to limit the scope of the present invention as described in greater detail herein. 1) Assemble column fittings on the second end of the column (and retaining means) and attach the column to the packing apparatus; 2) Make two particle support slurries in the packing solvent, one consisting of inert support particles, and the other containing the active support particles. For an immunoaffinity column, the packing solvent employed may be pH 7.0, 0.10 M potassium phosphate buffer and the slurry of the inert support particles typically may contain a diol-bonded material (e.g., 2 mg/mL diol-bonded silica). The second slurry contains the immunoaffinity support particles at a concentration that is determined by any generally known method (as set-forth in the examples below) and the desired thickness of the final active layer; 3) Begin flow of the packing solvent through the column. This is generally done at a rate of approximately 3 mL/min for immunoaffinity columns, but any rate may be employed. Make approximately five injections (at 150 μL per injection for a 1.0 cm long column) of the inert support slurry, followed by an increase in flow rate to approximately 5 mL/min for approximately 5 minutes; 4) Return the flow rate to approximately 3 mL/min and make the required number of injections of the active layer (as set-forth in the examples below). After making these injections, increase the flow rate to approximately 5 mL/min for approximately 5 minutes; 5) Return the flow rate to approximately 3 mL/min and make enough injections of the inert support slurry to fill the remainder of the column bed; 6) After the column bed has been filled, increase the column back pressure to the desired level, typically about 3000 to about 4000 psi. Allow the column to equilibrate at this pressure for approximately 10 minutes. Gradually release the pressure. Remove the column from the packing apparatus and place a frit (retaining means) and end fitting onto the open end of the column. The column is now ready for use.

The column, in addition to its relatively thin active layer, is also generally able to tolerate flow rates and pressures during sample injection that are capable of achieving the desired sample contact time with the active layer. The flow rate and pressure depends not only on the support particles employed in layer construction, but also on the column diameter and upper pressure limit that can be tolerated by the chromatographic system. In general, any flow rate and pressure necessary to achieve the desired residence time and tolerated by the chromatographic system employed is within the scope of the present invention. Typically, however, the columns may be subjected to flow rates of between about 0.01 to about 9.0 mL/min and pressures between about 10 to about 6000 psi. More preferably, the pressure is between about 100 to about 1500 psi.

II. Determination of the Free Analyte Fraction

Encompassed in the method of the invention is a means to determine the concentration of the free analyte fraction in a sample comprising a free fraction and a bound fraction. The method entails generating a calibration curve comprising data obtained by analyzing a series of standards containing a known concentration of the same analyte present in the sample. The concentration of the free analyte present in the sample, as described in detail below, is then determined by comparison to the calibration curve. Applicants have found that accurately determining the concentration of free analyte in a sample preferably involves the rapid and selective extraction of this fraction from the sample before significant release from its bound fraction. Additionally, applicants have found that this release generally occurs within a few seconds, particularly when the analyte binds a serum protein or other binding compound, such as human serum albumin or alpha 1-acid glycoprotein. The affinity columns described in detail above, therefore, are particularly suited for the method of the invention because they are capable of selectively extracting the free fraction within the millisecond time domain. The bound fraction on the other hand, does not bind significantly to the affinity column and is therefore present in the liquid fraction that passes through the column.

Accordingly, the method of the invention encompasses applying a sample to the affinity column (described in detail above) under conditions sufficient to bind the free analyte fraction without significant interference from its bound fraction, which passes through the column without adsorbing. The method, irrespective of the embodiment, also entails applying a series of standards to the same affinity column. "Standard" as utilized herein, shall mean a mixture that contains a known concentration of the analyte. The standard will preferably comprise the same analyte as is being detected in the sample and depending upon the embodiment of the invention, may also comprise a binding compound. "Series of Standards" as utilized herein, shall mean applying from about two to about five standards with different analyte concentrations to the column. In another embodiment of the invention, the series of standards may be determined without applying the standards to the column for each application of the method, such as when the calibration curve has been determined previously (from analysis of the same standard in an earlier test) or when the standard comes as a part of a kit. The series of standards will preferably contain concentrations of free analyte that are substantially comparable to the concentration of free analyte expected to be present in the sample.

In accordance with the method of the invention, the concentration of free analyte present in each standard is determined. This concentration may be determined based upon mass or volume measurements in which a known concentration of pure analyte is weighed and placed into a known volume of solution. Equally, a known volume of solution may be diluted or combined with another solution to prepare the final standard solution. In addition to these methods, the concentration may be determined by any means generally known in the art.

Additionally, the sample and standard, irrespective of the embodiment of the invention, are preferably injected onto the column under conditions that optimize a rapid and selective binding of the free analyte to the column. A number of conditions may affect the degree of free analyte binding to the column. These conditions are preferably optimized to achieve a high rate of binding and generally include: 1) an active binding agent in the column that is capable of binding the free analyte, 2) solution conditions (as set forth in more detail below) preferably favorable for binding to occur, 3) the binding capacity of the column is preferably equal to or greater than the amount of free analyte injected into the column (as discussed above), and 4) the residence time is typically optimized such that the time is preferably long enough that a significant concentration of the free analyte binds to the column and yet, short enough in duration to prevent significant dissociation from the bound fraction. The sample and standards may be applied to the column by any means generally known in the art, such as through the use of an injection valve or autoinjector system.

In addition to the parameters set forth above affecting binding capacity, a number of operating conditions employed during sample and standard injection onto the column are also preferably optimized. One such operating parameter is selection of the loading and elution buffers. The buffers selected preferably mimic the pH and solvent conditions of the sample to ensure that the equilibrium between the free and bound fractions is not disrupted. For example, when a biological sample is being analyzed, any physiological buffer, such as phosphate buffer, may be employed. And, the buffer typically will have a pH of approximately 7.2 to about 7.4 (pH of blood, serum or plasma). The temperature during injection of standard/sample onto the column is also significant. Again, the temperature will preferably mimic the natural temperature of the sample to avoid disrupting the binding properties of proteins in the sample prior to their application to the affinity column. Additionally, applicants have found that flow rate during sample injection can dramatically affect both extraction efficiency and dissociation of analyte from the bound fraction. Typically higher flow rates result in less dissociation, while slower flow rates increase extraction efficiency. Therefore, an intermediate flow rate is preferably employed during column injection and one generally skilled in the art of chromatography can readily determine this rate, which will vary depending upon the particular application. Typically, however, the flow rate is from about 0.1 to about 10.0 ml/min, and even more preferably, the flow rate is from about 0.5 to about 1.5 ml/min.

Accordingly, in one embodiment of the invention the concentration of free analyte present in the sample is determined by analyzing data from a series of standards comprising known concentrations of the same free analyte present in the sample without the presence of any binding compounds. In this embodiment, therefore, the concentration of the free fraction is determined by directly analyzing data from the free fraction of the series of standards ("Direct Method"). The sample, as detailed above, is applied to the column employing the same operating parameters as with injection of standard onto the column. The column separates the sample and series of standards into a free fraction and a bound fraction in the millisecond time domain. As detailed above, the free fraction of both the standard and sample is adsorbed to the column, while the bound fraction passes through the column.

The free analyte fraction of both the standard and sample isolated by the column is typically then detected as a signal by any means generally known in the art of analytical chemistry. "Signal" as utilized herein, shall mean the chemical or physical response that allows the analyte to be detected (in either the standard or sample). The signal can either be generated by the analyte itself or generated by another compound that is linked to the analyte (e.g., the use of a labeled analog of an analyte to allow the unlabeled analyte to be detected). In addition, the signal is specific to the particular detection method employed. Any detection method may be employed; however, the detection method employed is preferably the same for both the standard and sample in order to generate a reliable calibration curve. In addition, preferable features of a detection method include accuracy, sensitivity, and linear response over a broad range encompassing at least physiologically-relevant ranges, a high signal to background ratio in samples, and ease of use requiring a minimum of manual manipulation. Detection may be performed by either an on-line or off-line method. An "on-line" method, as utilized herein, shall mean a method in which there is a direct coupling between isolation of the free fraction (via affinity chromatography) and its detection such that the isolated fraction is automatically transferred to the detection mechanism through an interface that connects the two systems. An "off-line" method, on the other-hand, as utilized herein, shall mean a method in which the isolated fraction is collected and then manually transferred to the detection mechanism. Suitable detection methods include immunoassay, mass spectrometry, gas chromatography, and detection based upon ultraviolet absorbance, fluorescence detectors, and electrochemical detectors. Preferably, however, the detection technique utilized will comprise an on-line method with direct detection in order to facilitate efficiency of such detection.

The calibration curve can then be generated after the isolation and subsequent detection of signal, as detailed above, of the standard with known concentrations of free analyte. The calibration curve comprises a graph depicting the concentration of free analyte present in each standard versus the signal detected for each concentration. Additionally, the plot can be generated either manually, with a spreadsheet (e.g.,

Lotus or Excel) or by employing any computer program generally known in the art for linear or non-linear regression.

The concentration of the free fraction present in the sample can then be readily determined utilizing the calibration curve delineated above by simply comparing the signal detected from the free analyte fraction separated from the sample with the array of signals depicted on the calibration curve. This direct comparison is possible because the curve depicts the signal for known concentrations of the free fraction (generated from the series of standards). Therefore, the concentration of the free fraction of the sample may be determined by comparing its signal to signal depicted in the calibration curve for the standards with known free analyte concentrations. In yet another embodiment, the concentration of the free analyte fraction of the sample is determined by analyzing data from a series of standards comprising known concentrations of the same free analyte present in the sample and a binding compound. In this embodiment, in contrast to the Direct Method detailed above, the concentration of the free fraction of the sample is determined by analyzing data from the bound fraction and a total fraction (described below) from the series of standards ("Indirect Method"). In this embodiment, similar to the embodiment delineated above, the sample and series of standards are applied to the affinity column employing the same operating parameters. The column separates the sample and series of standards into a free fraction and a bound fraction in the millisecond time domain. Additionally, the free fraction of both the standard and sample is adsorbed to the column while the bound fraction passes through the column. In the Indirect Method, in contrast to the Direct method, the bound fraction of both the sample and series of standards is retained for further analysis in the next step of the method.

However, also in contrast to the Direct Method, the Indirect method employs the use of an additional inert control column. In this embodiment, the same sample and series of standards applied to the affinity column described above are applied to an inert control column. The inert control column comprises a column constructed in all details like the affinity column discussed above except that the support particles in its active layer are not derivatized with a binding agent. For example, the inert control column and affinity column employed in this embodiment are the same size, are constructed from the same materials, and are operated under the same parameters (i.e., pressure and flow rate). However, because the inert control column is not derivatized with binding agent, the free analyte fraction is not separated from either the sample or series of standards. Instead, a total analyte fraction comprising the bound fraction and free fraction pass through the column. The total fraction is retained from both the sample and series of standards for further analysis in the next step of the method.

The signal of the bound fraction of both the sample and series of standards is then detected by any means generally known in the art of analytical chemistry, as described in detail above for the Direct Method. In addition, the signal of the total fraction of both the sample and series of standards is also detected in accordance with the procedures described above for the Direct Method.

III. Determination of Analyte Concentration by Chromatographic Immunoassay and Near Infrared Fluorescent Labels

Yet another aspect of the invention is a method to determine the concentration of an analyte or free analyte fraction by chromatographic immunoassay and use of near infrared fluorescent labels. A chromatographic immunoassay is a technique that typically employs either an antibody or antigen immobilized to a column to perform various types of assays for compounds in complex matrices. This method is particularly useful in determining trace analytes that are at concentrations below the detection limits of conventional methods. Chromatographic immunoassays overcome this problem, as described below, typically by using a labeled antibody or labeled analyte analog that makes analyte detection, even at low analyte concentration, feasible.

Accordingly, a method of the invention encompasses applying a sample to the immunoaffinity column under sufficient conditions so that the analyte is extracted from the sample in the millisecond time domain. The details concerning column construction and operating parameters are set forth in detail above. The concentration of analyte present in the sample is then detected by chromatographic immunoassay and displacement of NIR labeled analyte analog.

In a preferred embodiment, the chromatographic immunoassay employed is the competitive binding immunoassay. In this type of immunoassay, the analyte is incubated with a fixed amount of labeled analog in the presence of a limited amount of antibodies that bind both to the analyte in the sample and the labeled analog. Because there are only a limited amount of antibodies present, the analyte in the sample and labeled analog must compete for the binding sites that are available on the antibody. After this competition has occurred, the bound and free portions of the sample are separated by the immunoaffinity column and the amount of labeled analog in either portion is analyzed. As the amount of analyte in the sample is increased, the amount of labeled analog that will bind to the antibodies will decrease, giving rise to an indirect measure of the analyte concentration that was present in the sample. Suitable approaches to competitive binding chromatographic immunoassays include displacement immunoassays, simultaneous injection immunoassays and sequential injection immunoassays.

In one embodiment of the invention, the competitive immunoassay format employed is the displacement competitive binding immunoassay. In this technique, the immunoaffinity column is allowed to bind with the labeled analog, followed by the application of sample to the column. As the sample passes through the column, the unlabeled analyte in the sample will bind to any antibody regions that are unoccupied by the label due to local dissociation/reassociation. The result is that an amount of labeled analog is displaced from the column proportional to the amount of unlabeled analyte in the sample, thus allowing detection of analyte concentration present in the sample.

The analyte or free analyte present in the sample, as stated above, is typically then detected as a signal by any means generally known in the art of analytical chemistry. This signal can either be generated by the analyte itself or generated by another compound that is linked to either the analyte or an analog of the analyte (e.g., see the detection of phenytoin in the Example below). Typically, in an immunoassay the signal is generated by a compound that is linked to the analyte or an analog of the analyte. For example, as illustrated in the Examples below, this may involve the use of a labeled analog of an analyte to allow the unlabeled analyte to be detected. The method of the invention typically utilizes labels capable of producing light through fluorescent processes. Suitable labels possessing fluorescent properties include a class of fluorescent dyes which are excited in the near-infrared range, specifically with an excitation wavelength between about 670 and about 800 nm and an emission wavelength between about 670 and about 1000 nm. Suitable NIR dyes include IRDye 800 CW dye, IRDye 800 RS dye, IRDye 700 DX dye, or IRDye 78 dye. The method of the invention may also utilize labels capable of producing light through chemiluminescent processes, including firefly luciferin, acridinium ester, luminol, and peroxyoxylates. Any other label, however, generally known in the art may be used.

Measurement of signal may be performed by either an on-line or off-line method. Preferably, however, the detection technique utilized will comprise an on-line method with direct detection in order to facilitate efficiency of the detection.

In one preferred embodiment, near infrared fluorescent labels are used. Specifically a NIR cyanine fluorescent label is coupled to the analyte of interest to generate a labeled analyte analog. IRDye 800 CW is one preferred label selected from near infrared fluorescent labels for detecting labeled analyte displacement in the immunoassay. IRDye 800 CW has an excitation wavelength of 770 nm wavelength and an emission wavelength from 780-900. In this embodiment, the IR Dye 800 CW- labeled analog is detected by excitation of the dye at an excitation wavelength of 770 nm and an emission wavelength from 780-900. Detection can be automated by placing a detector downstream and in-line of the column.

The methods of the present invention may be employed to determine the concentration of an analyte in a sample. In addition, these methods are particularly suitable for determining the concentration of the free analyte fraction of any sample comprising a bound and free fraction, to the extent that the free fraction is capable of being separated from the sample, as described herein. A typical application for the method, however, is the clinical analysis of the free fraction of a hormone, drug, protein or other endogenous or exogenous agents present in biological samples. The method may also be employed for the pharmaceutical analysis of free drug levels or drug metabolite levels in biological samples. Equally, the method may be employed in toxicology studies to quantify the free fraction of organic compounds and/or inorganic compounds present in a sample. In a research setting, the method may be utilized to study the mechanism of protein binding processes.

The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. Even so, this detailed description should not be construed to unduly limit the present invention as modifications and variation in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

All publications, patents, patent applications and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent, patent application or other reference were specifically and individually indicated to be incorporated by reference.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The embodiments were chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated.

EXAMPLES

The following examples illustrate the methods of determining the concentration of free analyte in a sample as well as methods of preparing reagents and optimizing/characterizing conditions that are useful for determining the concentration of free analyte in a sample. The examples demonstrate certain methods and are not intended to limit the scope of the present invention. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

The examples illustrate the ability of the method of the present invention to detect the free fraction of phenytoin from a sample comprising phenytoin and human serum albumin ("HSA"). Phenytoin is an anti-epileptic drug that is known to have significant protein binding in blood.⁶'⁷ This drug is mainly bound in blood to the protein human serum albumin (HSA), with approximately 90% of phenytoin being complexed with this protein at normal therapeutic concentrations in adults. ^" Because of phenytoin's pharmaceutical significance and its binding affinity for HSA₅ it is an excellent model to illustrate the broad applicability of the method of the current invention.

The following description of the reagents, apparatus, and conditions are common to all examples, where relevant.

Reagents. The phenytoin (99% pure), mouse IgG, human plasma (lyophilized, pooled), and HSA (Cohn fraction V, 99% fatty acid free) were purchased from Sigma-Aldrich (St. Louis, MO). The IRDye 800 CW (iV-hydroxysuccinimide, or NHS ester) was donated by LI-COR Biosciences (Lincoln, NE). The monoclonal anti-phenytoin antibodies (clone P0825.1, purified from ascites fluid; stored in pH 7.2 phosphate buffered saline plus 0.05% sodium azide) were obtained from Accurate Chemical (Westbury, NY). Nucleosil Si-300 (7-μm particle size, 300-A pore size) was purchased from PJ. Cobert (St. Louis, MO). Reagents for the bicinchoninic acid (BCA) protein assay were from Pierce (Rockford, IL). The phenytoin calibration standards, free phenytoin calibrators, and control serum samples (pooled serum containing known amounts of phenytoin) were obtained from Abbott Laboratories (Chicago, IL). Other chemicals were reagent grade or better. All aqueous solutions were prepared using deionized water from a Nanopure water system (Barnstead, Dubuque, IA).

Apparatus. Mass spectra of phenytoin and its conjugates were obtained using electron ionization mass spectrometry (EI-MS) performed on a VG AutoSpec mass spectrometer from Fisons (now Waters/Micromass) (Milford, MA). ¹H NMR spectra were acquired for phenytoin and its conjugates on a Bruker DRX Avance 500 MHz spectrometer (Billerica, MA) using samples dissolved in deuterated dimethyl sulfoxide (DMSO). Ultrafiltration was performed using Centrifree Micropartition Devices (MW cutoff, 30 kDa; sample capacity, 0.15-1.0 mL) from Amicon (Danvers, MA), along with a fixed rotor basket centrifuge from Dynac (Parsippany, NJ). The temperature during the ultrafiltration experiments was controlled by placing the centrifuge in a Precision P3 incubation cabinet from Expotech (Houston, TX). Samples for the BCA protein assay were analyzed using a Shimadzu UV 160U absorbance spectrophotometer (Kyoto, Japan). The fluorescence properties of the labeled phenytoin and serum samples were examined using a QuantaMaster spectrofluorometer from Photon Technology International (Rockwood, TN), using an excitation wavelength of 770 nm and an emission wavelength range of 780-900 nm. The HPLC system used to analyze free phenytoin fractions after ultrafiltration consisted of a Jasco PU980 pump (Easton, MD), an Alltech water jacket, and a LDC Analytical 3100 UV detector (Riviera Beach, FL), along with a 2.1 mm i.d. x 4.5 cm HSA column (prepared as described previously).⁸'³⁵ Samples were injected onto this system using a 20-μL sample loop and a Thermoseparations AS3000 autosampler (Schaumberg, IL). The same chromatographic system was used to determine the extraction efficiency of the anti -phenytoin immunoextraction columns, with the column or a control column being used in place of the HSA column. These immunoextraction columns were packed according to a previously published method.³⁶'³⁷

The chromatographic system used for the UFIDA method was similar to that described in the previous paragraph but included a second Jasco PU980 pump and a Rheodyne model EV700 six port switching valve (Cotati, CA) for alternating passage of the application and elution buffers through the anti-phenytoin immunoextraction column. The labeled phenytoin was injected onto this system in volumes ranging from 5 to 100 μL, followed by 5-μL injections of the sample. Detection of the labeled phenytoin was performed using a custom-built HPLC NIR fluorescence detector from LI-COR. This detector was constructed with a 25-μL flow cell positioned at the interface of the laser and detector focus, using an optical system³⁸ with the laser diode source and microscope detector being positioned 90° with respect to the flow cell. The emitted wavelength of the laser diode was 785 nm. Excitation wavelengths were selected using a 20-nm band-pass filter centered at 820 nm. The temperature of the immunoextraction column or control column was maintained by placing these into water jackets (Alltech) and by coupling these jackets to a Brinkmann circulating water bath (Westbury, MA). All chromatographic data were collected using programs written in Labview 5.1 (National Instruments, Austin, TX). Retention times and areas of the resulting chromatographic peaks were calculated using PeakFit 4.12 (Systat Software, Richmond, CA).

Example 1

Preparation of labeled phenytoin. Phenytoin was converted into an amine derivative (i.e., 3-7V-amino-5,5- diphenylhydantoin, or ADPH) by the reaction scheme shown in Figure 5.^39"42 A mixture of 2.50 g phenytoin (0.01 mol) and 3.90 mL hydrazine hydrate (0.08 mol) was heated at 135⁰C for 5 h; no other solvent was required for this reaction. ADPH was recrystallized by placing 1 g of the solid product in approximately 15 mL ethanol. Deionized water was then added, and this mixture was allowed to stand undisturbed at 4⁰C for two (2) days. The resulting crystals were filtered and dried at 80⁰C under vacuum for 8 h.

Analysis of the product of this reaction by EI-MS yielded a molecular ion with a mass-to-charge ratio of 267.1019 (mass accuracy, 4.2 ppm) and fragment ions with masses in agreement with those previously reported for ADPH.³⁹ A ¹H NMR spectrum for this product also agreed with that expected for ADPH, giving a singlet peak at 5 ppm (approximate relative area, 1.40) for the NH₂ group, a group of peaks at 7.4 ppm (relative area, 10) for the aromatic protons, and a singlet peak at roughly 9.4 ppm (approximate relative area, 0.72) for the proton on the second nitrogen in the hydantoin ring of ADPH. A singlet was also observed at 9.8 ppm (relative area, 1.10), the result of abyproduct (i.e., 5,5-diphenyl-l,2,4-triazine-3,6-dione) which was estimated to make up approximately 40-50% of the final product when analyzed by HPLC.

The ability of this ADPH preparation to act as a nucleophile was initially tested by combining a portion of this product with dansyl chloride to form dansyl hydrazide derivative.⁵⁸ This reaction was performed by placing 0.1466 g (0.5 mmol) of dansyl chloride in 4 mL of pH 4.0, 25 mM sodium acetate buffer and adding 0.1418 g (approximately 0.5 mmol) of the ADPH product in 1 mL DMSO. The result was a yellowish-brown precipitate, as would be expected for a dansyl hydrazide derivative.

ADPH was conjugated to NHS-activated IRDye 800 CW dye by using an approach similar to that shown in Figure 5.⁴³ A solution of 20 μmol ADPH in 0.5 mL DMSO and 1 mL of pH 8.0, 0.10 M potassium phosphate buffer was slowly added to a solution of 9 μmol of NHS-activated IRDye 800 CW dye in 0.5 mL DMSO. The resulting mixture was slowly stirred with a magnetic stir bar for 4 h in an ice water bath. This mixture was then dried to remove all solvent, with the remaining residue being dissolved in 10 mL of pH 7.4, 0.067 M potassium phosphate buffer. This product was extracted three times with 10 mL portions of ethyl acetate to remove any unconjugated reactants. The remaining aqueous fraction was dried to remove any ethyl acetate yielding a residue containing the phenytoin-dye conjugate and unconjugated dye. No further purification of the final labeled phenytoin dye conjugate (i.e., labeled phenytoin) was required for this study.

The purity of the labeled phenytoin in the final product was estimated to be 62%. This purity was sufficient for use in this study, since an excess of labeled phenytoin was used in the displacement assay. It was found later that the 38% of unconjugated dye in this preparation did not create any noticeable interference due to background signal or nonspecific binding in the final UFIDA assay. The purity of this labeled phenytoin was measured by comparing the NIR fluorescence for the retained peak

(i.e., the labeled phenytoin) and non-retained peaks (containing the unconjugated dye) when a 5-μL sample of this preparation was injected in pH 7.4, 0.067 M potassium phosphate buffer at 0.5 mL/min onto a 2.1 mm i.d. x 4.5 cm immobilized HSA column, with pH 7.4, 0.067 M potassium phosphate buffer also being used as the mobile phase. This HSA column was prepared as described previously⁸'³⁵'⁴⁴'⁴⁵ and had a protein content of 28 (+ 2 mg) HSA/g silica (± 1 SD), as determined by a BCA protein assay. As has been noted for phenytoin⁸'³ this HSA column was found to retain the labeled phenytoin while the free acid form of the NIR fluorescent dye eluted near the column void volume of this column. The phenytoin-dye conjugate was also strongly retained by anti-phenytoin antibodies.

A stock solution was made for the labeled phenytoin in pH 7.4, 0.067 M phosphate buffer. The concentration of labeled phenytoin in this stock solution was estimated to be 558 (+ 20) μM after a correction was made for unconjugated dye in the final product. This concentration was determined by using the HPLC assay results of this conjugate's purity and fluorescence of this stock solution versus standards containing known concentrations of the NIR fluorescent dye. When not in use, this stock solution was stored in the dark in an amber vial at 4⁰C. This stock solution was used over the course of approximately four months during this study.

Example 2 Preparation of immunoextraction column.

Prior to immobilization, the anti-phenytoin antibodies were transferred from their original solution (pH 7.2 phosphate buffered saline containing 0.05% sodium azide) into pH 6.0, 0.10 M potassium phosphate buffer. This transfer was accomplished by applying a 3.0-mL sample of these antibodies to a 10-mL Econo-PAC 10DG column (exclusion limit, 6,000 Da) from BioRad (Hercules, CA) using pH 6.0, 0.10 M potassium phosphate buffer as the mobile phase. The collected antibodies were stored at 4⁰C in the same pH 6.0, 0.10 M phosphate buffer.

Prior to its use in immobilization, Nucleosil Si-300 silica was converted into a diol- bonded form according to a previous method.⁴⁴ The final diol coverage of this material was 306 (+ 3) μmol/g silica, as determined in triplicate by an iodometric capillary electrophoresis assay.⁴⁵ The anti-phenytoin antibodies in pH 6.0, 0.10 M potassium phosphate buffer were immobilized onto this diol-bonded silica by the Schiff base method.⁴⁶ An inert control support was prepared in an identical manner but with no antibodies being added during the immobilization step. This type of support was found to be an adequate control for this work because of the specificity of the anti-phenytoin antibodies and relatively low nonspecific binding of the analytes or labeled analogues to the control support; however, a control support containing immobilized nonspecific antibodies could also be used. After immobilization, the anti-phenytoin support was washed three times with pH 7.4, 0.067 M potassium phosphate buffer and stored at 4°C until use. A portion of this support was washed several times with deionized water to remove any salts from the buffer, dried, and analyzed in triplicate by using a BCA protein assay.⁴⁷ This assay gave a protein coverage of 29 (+ 7) mg of antibodies/g of silica when using mouse IgG as the standard and the control support as the blank.

The anti-phenytoin support was used to prepare immunoextraction columns,²'³⁷ which had an inner diameter of 2.1 mm and a total length of 0.5 cm. The central layer of these columns was approximately 1 mm thick (940 μm) and contained the anti- phenytoin support, while the remainder of the columns contained the inert control support. These columns were prepared by making fifteen 50 μL injections at 3 mL/min of a 4.2 mg/mL slurry of the control support to one end of the column in the presence of pH 7.4, 0.067 M phosphate buffer. This was followed by application of the same buffer at 5 mL/min for 5 min to stabilize this layer to a thickness of approximately 0.20 cm. A small layer of the anti-phenytoin support was next placed within this column by making fourteen, 50-μL injections of a 2.1 mg/mL slurry of this material in pH 7.4, 0.067 M phosphate buffer and at 3 mL/min; this layer was also stabilized by later increasing the flow rate to 5 mL/min for 5 min. The remainder of this column was filled in the same manner with the inert control support.

The initial concentration of each support slurry used in packing the immunoextraction column was determined by turbidity measurements made at 800 nm versus standard solutions containing known concentrations of the control support in pH 7.4, 0.067 M phosphate buffer. The immunoextraction columns were stored in pH 7.4, 0.067 M phosphate buffer at 4⁰C when not in use. The typical back pressure of these columns at 0.5 to 1.6 mL/min was 130 to 420 psi (0.9 to 2.9 MPa), and increased by only 19% over four months of regular use.

Example 3 Characterization of immunoextraction column.

The amount of active anti-phenytoin antibodies in the immunoextraction columns was determined by frontal analysis.⁴⁷'⁴⁸ This was performed using solutions that contained 2 to 40 μM phenytoin in pH 7.4, 0.067 M phosphate buffer and that were applied at 1.2 mL/min. The breakthrough curves for phenytoin in these solutions were monitored at 205 nm, with all runs being conducted in triplicate at 37°C. Elution of the retained phenytoin was later accomplished by applying pH 2.5, 0.067 M phosphate buffer to the immunoextraction columns. Sample application and column regeneration were performed by using pH 7.4, 0.067 M phosphate buffer as the mobile phase. Corrections for the system void time and non-specific binding of phenytoin to the column were made by conducting identical experiments using a column with the same dimensions as the immunoextraction column but containing only the control support. The extent of phenytoin binding to this control support was less than 8% of the total binding measured for the immunoextraction column.⁸'³⁵

The extraction efficiency of the immunoextraction column was determined by making 20-μL injections of a 6 μM phenytoin standard at 37°C in pH 7.4, 0.067 M phosphate buffer; these injections were made onto both a column containing only the control support and onto the immunoextraction column. The amount of non-retained phenytoin was measured on each column at 205 nm and at flow rates ranging from 0.6 to 1.6 mL/min. Similar experiments were conducted when no column was present. The difference between the total peak areas measured with the control column and with no column present was less than 3%. A comparison of these areas to the non- retained areas measured for phenytoin on the immunoextraction column under the same conditions allowed the fraction of extracted phenytoin to be calculated at each flow rate.

Example 4

Ultrafiltration of phenytoin samples. Ultrafiltration was used as a reference method for validating the free fraction measurements made for phenytoin in this study.⁴'¹⁹'²¹'⁴⁹ Ultrafiltration was performed on control serum samples containing known concentrations of phenytoin and on various HSA/phenytoin samples containing 550-750 μM HSA and 30-50 μM phenytoin in pH 7.4, 0.067 M potassium phosphate buffer. Each of these samples was centrifuged in the presence of an ultrafiltration membrane at 37°C for 45 min at 1,500 x g. After centrifugation, approximately 0.5 mL of the filtrate was collected and stored at 4°C until further use.

The concentration of free phenytoin in the filtrate was measured by using HPLC along with the same HSA column described earlier for examining the purity of the labeled phenytoin. Samples containing 20 μL of the filtrate were injected onto this column in triplicate at 0.5 mL/min and at room temperature. The elution of phenytoin was monitored at 205 nm and gave a retention factor of 2.3 on this column. No peaks from other sample components were noted in the vicinity of the retention time for phenytoin. A linear response was obtained on this column for phenytoin standards containing 2 to 15 μM phenytoin in pH 7.4, 0.067 M phosphate buffer (correlation coefficient, 0.9998 for n = 5). A calibration curve prepared from these standards was then used with the measured peak areas to determine the free concentration of phenytoin in the filtrate samples.

Non-specific binding by phenytoin in the ultrafiltration device was measured by using a series of standards that contained phenytoin in pH 7.4, 0.067 M phosphate buffer. The amount of phenytoin in the recovered filtrate was then determined by HPLC, as described previously. These experiments indicated that phenytoin had 7.6 (+ 0.1)% nonspecific binding to ultrafiltration membrane under the conditions used in this assay, in agreement with results of earlier studies.¹⁹ All phenytoin results obtained by ultrafiltration were later corrected for nonspecific binding based on this value.

Example 5

Ultrafast immunoextraction/displacement assay.

The labeled phenytoin and immunoextraction column developed in this work were used in an UFIDA method according to the scheme given in Figure 4 A. At the beginning of this assay, 20 μL of a 55.8 μM sample of the labeled phenytoin was applied to the immunoextraction column at 0.8 mL/min using pH 7.4, 0.067 M potassium phosphate buffer as the mobile phase. Once a baseline had been established, the flow rate was increased to 1.2 mL/min. A 5 μL sample containing a phenytoin/HSA mixture or phenytoin in human serum was injected at 6 min after application of the labeled phenytoin. Once the resulting displacement peak had eluted, the retained phenytoin and any remaining labeled phenytoin were removed from the column by applying a pH 2.5, 0.067 M phosphate solution as the elution buffer at 1.2 mL/min for 5 min. The column was then regenerated by applying pH 7.4, 0.067 M phosphate buffer at 0.8 mL/min for 5 min prior to the next application of the labeled phenytoin. All experiments measuring free phenytoin concentrations on this system were performed in triplicate at 37°C.

Example 6

Selection of conditions for ultrafast immunoextraction.

An underlying requirement for free drug measurements by ultrafast immunoextraction is that this method must be able to extract a representative portion of a drug's free fraction while avoiding any appreciable release of the same drug from its bound form in the sample. As has been shown previously for other analytes, the timescale needed for this process can be estimated by using the dissociation rate constant of a drug from its carrier agents in a sample along with the extent of this binding in typical samples.¹'²'³⁶

Several previous reports have examined the binding of phenytoin to HSA, the major binding agent for this drug in blood or serum.⁸'³⁵ Two specific regions on HSA that are involved in this binding are the indole-benzodiazepine site (Sudlow site II) and the digitoxin site of HSA. These sites have been reported to have association equilibrium constants for phenytoin of 1.04 x 10⁴ and 6.5 x 10³ M^"1, respectively, at pH 7.4 and 37⁰C. It has also been found that phenytoin has allosteric plus possible direct interactions with the warfarin-azapropazone site (Sudlow site I) and tamoxifen site of HAS.⁸'³⁵ The combined result of these interactions is a bound fraction for phenytoin that makes up 80 to 90% of its normal therapeutic levels in serum or blood.^8'14'³⁵ Through previous kinetic measurements using HSA columns, the overall dissociation rate constant for phenytoin from these binding sites of HSA has been estimated to be 10.8 (± 0.05) s^"1 at pH 7.4 and 37⁰C.⁵⁰ Based on this result and previous work performed in the ultrafast immunoextraction of warfarin and thyroxine,¹'² it was originally estimated that the analysis of free phenytoin fractions by UFIDA would require a sample residence time in an immunoextraction column of less than 200 ms. To obtain these conditions, a sandwich column was used that contained a 2.1 mm LD. x 940 μm thick layer of an anti-phenytoin support. This gave an expected residence time for samples in the immunoextraction layer of roughly 100 ms at a flow rate of 1.2 mL/min.

Frontal analysis was used to estimate the binding capacity of the anti-phenytoin antibodies in this immunoextraction column. This gave a total binding capacity of 67 (+ 3) pmol phenytoin at pH 7.4 and 37⁰C. It was further found from protein assays that this column contained approximately 290 (+ 90) pmol antibodies. This gave an effective concentration of active antibody binding sites in the immunoextraction layer of 3.4 (± 0.2) μM and a relative activity of the antibodies for phenytoin of 12 (+ 0.1)% (assuming there were two accessible binding sites per antibody). Although this relative activity was low, the total column binding capacity was sufficient for this work since it was roughly two times higher than the amount of free phenytoin that would be expected at the high end of this drug's therapeutic range in a 5 μL sample (i.e., based on a 90% bound fraction and a total phenytoin concentration of 80 μM). If desired, this relative activity and binding capacity could be improved by employing more site-selective techniques for antibody immobilization; examples include the use of hydrazide-activated supports plus antibodies that have been oxidized in their carbohydrate regions, or the use of biotin tags in these same regions along with avidin or avidin-containing supports.^51"53

The association equilibrium constant for phenytoin with the immobilized anti- phenytoin antibodies was determined by frontal analysis to be 5.4 (+ 0.3) x 10⁸ M^"1 at pH 7.4 and 37⁰C (Note: no significant change in this value was noted at lower flow rates). Although this result represents reasonably strong binding, this association equilibrium constant is ~20-fold lower than a solution-phase equilibrium constant of 1 x 10¹⁰ M^"1 that was provided by the manufacturer of these antibodies. This difference is not surprising because immobilized antibodies can often have lower binding constants than their soluble forms.⁵⁴'⁵⁵ Using this equilibrium constant and the measured binding capacity of the immunoextraction column, the retention factor for phenytoin on this column at pH 7.4 and 37⁰C was estimated to be 348 (+ 26). This value was calculated by using the relationship k = K_A mi/ FM, where K_A is the association equilibrium constant for phenytoin with the antibodies, mi is the moles of active antibodies in the column, Vu is the void volume of the immunoextraction layer, and (M]JV_M) is the effective concentration of active antibodies in this layer.⁴⁸ Further examination of the frontal results, as described in,²⁸ gave a measured association rate constant (k_a) for phenytoin with the immobilized anti-phenytoin antibodies of 2.4 (+ 0.4) x 10⁶ M^'1 s^"1 at pH 7.4 and 37⁰C. From the relationship K_A = kJh, the dissociation rate constant (Je_t) for phenytoin from the immobilized antibodies was also determined, giving a value of 4.4 (+ 0.8) x 10^"3 s^"1. All of these results indicated that any phenytoin extracted by the immobilized antibodies would bind tightly to this column and only slowly dissociate from these antibodies in the presence of a pH 7.4 buffer.

The ability of this column to extract phenytoin was measured by comparing injections of phenytoin standards made onto this column versus injections made onto a column of identical size but containing only an inert control support. Table 1 summarizes the results that were obtained. These experiments were performed at flow rates that gave residence times in the range of 78-207 ms for samples as they passed through the antibody layer of the column. The concentration and quantity of phenytoin that was injected (20 μL of a 6 μM solution, or 120 pmol phenytoin) corresponded to the free amount of this drug that would be expected in serum at typical therapeutic levels. It was found that 95% or more of the phenytoin was extracted by the column when using sample residence times of 100 ms or greater in the immunoextraction layer, and 98% was extracted when using residence times of 150 ms or greater in the immunoextraction layer. These results are in agreement with previous observations made for the ultrafast immunoextraction of thyroxine and warfarin.¹'² The immunoextraction column was also used in studies examining the effect of flow rate and sample residence on the apparent free fraction that was obtained for phenytoin in a displacement assay. It was found that a flow rate of 1.2 mL/min or greater (i.e., a sample residence time in the antibody layer of 103 ms or less) gave a consistent measured free phenytoin fraction of 15.8% for the test mixture used in Table 1. However, slower flow rates and longer sample residence times resulted in a greater apparent free fraction (1.9% higher at 0.8 mL/min and 5.7% higher at 0.6 mL/min). As indicated in previous simulations performed for warfarin and thyroxine,¹'² this increase in the apparent free fraction is believed to reflect dissociation of the analyte from proteins or other binding agents in the sample. Thus, as a compromise between extraction efficiency and accuracy, an injection flow rate of 1.2 mL/min (i.e., a sample residence time of roughly 100 ms in the immunoextraction layer) was used in all later experiments for free phenytoin measurements.

Table 1. Ultrafast immunoextraction of phenytoin at various flow rates in a sample containing a fixed total amount of phenytoin and HSA^a

Residence time in antibody

Flow rate layer of immunoextraction Extraction Measured free fraction (mL/min) column (ms) efficiency (%) phenytoin (%)

0.6 207 100 (±10) 16.7 (±0.6) 0.8 155 98 (±2) 16.1 (±0.5) 1.2 103 95 (±3) 15.8 (±0.5) 1.4 89 92 (±3) 15.8 (±0.4) 1.6 78 87 (± 5) 15.8 (±0.2)

^aThese values were determined using sample that contained 550 μM HSA and 35.0 μM phenytoin. The numbers in parentheses represent a range of ±1 SD. Example 7

Behavior of NIR fluorescent label under assay conditions.

The next item considered was the signal intensity and behavior of the labeled phenytoin-dye conjugate under the conditions to be used for ultrafast immunoextraction. It was found in pH 7.4, 0.067 M phosphate buffer that the NIR fluorescence of the labeled conjugate gave a linear response over a broad range of concentrations. This linear range extended from approximately 1.7 fmolto 2.1 pmol (i.e., 0.33 to 412 nM for a 5 μL injection, with a lower limit of detection of 1.4 nM at a signal-to-noise ratio of three). Although this amount is less than the free phenytoin levels in 5 μL of serum (i.e., 20 to 40 pmol or 4 to 8 μM), this UFIDA format only requires that a small, representative amount of the labeled phenytoin be displaced by an injected analyte. Thus, the response for the NIR fluorescent label was more than adequate for analyzing the displacement peaks that were created during the detection of free phenytoin fractions in such samples.

Another item examined was the effect of biological factors on the fluorescence of the labeled phenytoin-dye conjugate. This was of interest since it has been noted in previous work with chemiluminescent labels that up to a 30% change in signal can be seen for a labeled analyte in the presence of serum versus buffer.¹ To study this effect for the labeled phenytoin-dye conjugate, the emission spectrum of this labeled phenytoin-dye conjugate was obtained in the presence and absence of human plasma (i.e., serum plus clotting factors), as shown in Figure 6A. This information was acquired using a 100 μM solution of the labeled phenytoin-dye conjugate in 1 mL of pH 7.4, 0.067 M phosphate buffer or serum. It was found that there was no appreciable reduction in signal intensity (i.e., less than 3% change) between a buffered standard and plasma containing the labeled phenytoin-dye conjugate. Similar results were obtained when comparing plasma and buffered solutions containing 0.33 to 412 nM labeled phenytoin, which gave less than a 17% difference in signal at all concentrations examined. The similarity of the plasma and buffer results indicated that there was either only a small amount of binding between the labeled phenytoin and HSA in plasma or that this binding did not have any appreciable affect on the NIR fluorescence of the dye in this conjugate.

The actual extent of interferences from the sample matrix would be expected to be even smaller than 3 to 17% when the labeled phenytoin-dye conjugate is used in a displacement assay, since the peak for the displaced conjugate elutes after the non- retained fraction of the sample. This is demonstrated in Figure 6B, where even the highest concentration phenytoin standards gave displacement peaks with a mean elution time that occurred 1 min after the non- retained components of a serum sample (as represented by HSA). The overlap of the sample and displacement peaks was estimated to be less than 5% for this assay, which would further reduce any effects of the sample matrix on the fluorescent signal of the NIR dye used in the labeled phenytoin conjugate.

The emission spectrum for a sample of human plasma with no NIR dye added was acquired to see what type of background signal could be expected from such a sample. This spectrum is also shown in Figure 6A and gave no detectable signal at the emission wavelengths that were monitored. From this result, as well as the difference in sample and displacement peak elution times noted in Figure 6B, it was concluded that no significant background signal should have been present under the conditions used in this study for detection of the labeled phenytoin-dye conjugate in the displacement peaks.

Example 8 Optimization of UFIDA method.

After the initial conditions for ultrafast immunoextraction and detection of the labeled phenytoin-dye conjugate had been selected, these components were combined and optimized for use in an UFIDA assay for measuring free phenytoin fractions. One item considered was the effect of varying the amount of labeled phenytoin that was applied to the system for analyte detection. This item was studied by applying 5 to 100 μL of a 0.558 to 55.8 μM solution of the labeled phenytoin-dye conjugate (i.e., 2.8 pmol to 5.6 nmol) at 0.8 ml/min to the immunoextraction column prior to the injection of a 5 μL sample of 35 μM phenytoin (i.e., a typical therapeutic concentration expected for free phenytoin in serum). The amount of labeled phenytoin used under these conditions ranged from 0.04- to 84-times the binding capacity of the immunoextraction column. The area of the displaced peak gave less than a 6% change when the amount of labeled phenytoin was at least 110 pmol (e.g., a 20 μL injection of 5.58 μM labeled phenytoin), or conditions in which the amount of the labeled phenytoin-dye conjugate was present in more than a 1.6-fold excess versus the column binding capacity.

The only change noted when using larger amounts of labeled phenytoin was a slight increase in the time it took to wash the excess, non-retained labeled conjugate from the column. When using small amounts of this conjugate, it took approximately 3 min to remove 99.9% of the non-retained labeled phenytoin from the column. However, it took around 5.5 min to wash off the excess conjugate when using the highest amounts of labeled phenytoin that were examined in this work. Based on these results, a 20 μL injection of a 5.58 μM preparation of the labeled phenytoin-dye conjugate (i.e., 110 pmol) was used along with a wash time of 3 min in the final UFIDA method as a compromise between assay speed and response.

Another item considered in developing the UFIDA method was the effect of varying the time between injection of the labeled phenytoin and the injection of a sample. This was examined to see if overlap of the sample peak with the remaining non- retained phenytoin-dye conjugate (at small injection times) or loss of the retained phenytoin-dye conjugate (at long injection times) gave a significant change in response for the displacement peak. The results of these studies are shown in Figure 7 A for a column loaded with 20 μL of a 5.58 μM solution of labeled phenytoin-dye conjugate at 0.8 mL/min, followed by the injection of a 4 μM phenytoin sample at 1.2 mL/min and at various times after application of the labeled conjugate (Note: the flow rate was changed to 1.2 mL/min after 1.5 min into each run). Using the displacement peak obtained with a sample injection at 2 min as the reference, the changes in area noted for displacement peaks measured after sample injections at 4, 6 or 12 min were 1.7, 1.9, and 4.8% respectively. It was concluded from these data that sample injection should be performed in 2 to 6 min after application of the labeled phenytoin- dye conjugate to give less than 2% variation in the displacement peak's size. These conditions minimized loss of the retained labeled phenytoin while also allowing sufficient time for excess labeled conjugate (and any associated contaminants) to be washed from the column.

The next study considered the use of sequential sample injections during the UFIDA method. This was done to determine whether it was possible to perform more than one analysis per column loading of the labeled phenytoin-dye conjugate. In these experiments four 5 μL injections were made of a sample containing 35 μM phenytoin plus 550 μM HSA. These samples were injected at 6, 9, 12 and 14 min after applying 20 μL of 5.58 μM labeled phenytoin-dye conjugate to the system. As shown in Figure 7B, the use of two sequential sample injections gave less than a 5% change in displacement peak area and less than a 3% change in peak height. The intensity of the displacement peaks then began to decrease with further sample injections, giving signals that were 36 and 93% lower (versus the first sample) for the third and fourth injections. In the remainder of this study only one sample injection was performed after each application of the labeled phenytoin-dye conjugate. However, the findings in Figure 7B indicate that multiple injections could be used in such an assay to further increase sample throughput in the UFIDA method.

Elution of the retained phenytoin and labeled phenytoin-dye conjugate in the UFIDA method was accomplished by using a pH 2.5, 0.067 M phosphate buffer applied at 1.2 mL/min for 5 min. Regeneration of the immunoextraction column was conducted by applying pH 7.4, 0.067 M phosphate buffer for at least 5 min prior to a new injection of the labeled phenytoin-dye conjugate. The UFIDA method was found to be quite stable under these conditions, allowing a reproducible response to be obtained on a single column over at least 250 injections and four months of regular use.

Example 9

Validation of UFIDA method. The final conditions used in the UFIDA assay for free phenytoin measurements are summarized in Example 5. An example of a typical run obtained under such conditions is presented in Figure 4B. The displacement peak for this assay appeared within 2-3 min of sample injection, and the total assay time (i.e., one injection, elution and regeneration cycle) was 20 min. However, it was found that the total time to detect and analyze free phenytoin fractions could be reduced to less than 10 min per sample by using multiple sample injections after each application of the labeled phenytoin (see previous section).

A typical calibration curve obtained for the UFIDA method is shown in Figure 8. The lower limit of detection for this method was 570 pM phenytoin (S/N - 3) for a 5 μL sample (i.e., 2.9 fmol). The linear range (i.e., the range of analyte concentrations giving a response within 10% of the best- fit line) extended from the lower limit of detection up to approximately 10 μM phenytoin (approximately 50 pmol). This linear response covered the entire range of free phenytoin concentrations that would be expected at normal therapeutic levels of this drug. The calibration curve for this assay did level off as phenytoin concentrations above 10 μM were injected. This behavior is probably due to saturation of the immunoextraction column, since these high sample concentrations gave rise to amounts of phenytoin that approached or exceeded the binding capacity of this system (i.e., 50 pmol phenytoin in 5 μL of a 10 μM sample versus a 67 pmol binding capacity for the column).

The precision of this assay was determined by making replicate injections of standards, phenytoin/HSA mixtures and spiked control serum samples. A relative standard deviation of ±0.5% or less was seen for standards containing free phenytoin concentrations of 1.24 nM to 2.02 μM. As shown in Example 10, the precision was about 2% to 5% for serum and HSA samples that contained phenytoin at typical therapeutic concentrations.

The accuracy of the UFIDA assay was first assessed by comparing it to that for ultrafiltration in the analysis of phenytoin/HSA mixtures that had been prepared in pH 7.4, 0.067 M potassium phosphate buffer. Table 2 summarizes the results that were obtained. The sample concentrations used were chosen to match the levels of HSA and phenytoin that would be expected in serum samples containing typical therapeutic concentrations of phenytoin. These samples gave average free phenytoin fractions of 15.9, 13.7 and 12.0% at HSA concentrations of 550, 650 and 750 μM, respectively (i.e., results similar to literature values reported for free phenytoin fractions in serum).¹¹'¹⁴ All of the twenty- five phenytoin/HSA samples that were examined gave statistically identical results at the 95% confidence level for UFIDA versus ultrafiltration. In addition, all of the UFIDA and ultrafiltration results overlapped within 1 SD of their values and had differences in these values of only 1.6% to 5.4% (average, 2.9%). Although there was a small apparent bias in the ultrafiltration results compared to UFIDA, the size of this bias was strongly linked with experimental uncertainly in the nonspecific binding measured for the ultrafiltration membranes.

Another comparison was made between UFIDA and ultrafiltration in terms of their ability to determine free phenytoin concentrations in human serum. This comparison was conducted with standards and samples that are used in a commercial immunoassay kit for free phenytoin measurements.¹²'¹⁵'²¹ The four standards supplied with this kit (total phenytoin concentrations, 1.98 to 12 μM in phosphate buffer) gave a linear response for the UFIDA method, with a best fit line of y = 4.85 (+ 0.04) x + 2.9 (+ 3.0) and a correlation coefficient of 0.9932 (n = 4). Serum samples containing total phenytoin concentrations of 10 to 40 μM gave the results shown in Table 3. Comparison of the UFIDA and ultrafiltration results for these samples again resulted in statistically identical values for the measured free fractions of phenytoin. When using ultrafiltration, control sera containing 10 to 40 μM concentrations of total phenytoin gave free phenytoin concentrations of 1.32 to 6.11 μM (or 13.4 to 15.4% free fractions), while analysis of the same samples using UFIDA gave free phenytoin concentrations of 1.27 to 5.99 μM (or free fractions of 12.2 to 15.1%). The difference between the results of these two assays was less than 2 to 5%, with all differences being within 1 SD of the measurements. These results supported the conclusion that there was good agreement between the UFIDA and the ultrafiltration reference method. Example 10

Comparison of UFIDA and ultrafiltration.

Although UFIDA and ultrafiltration gave comparable results for the samples in Tables 2 and 3, they did differ in terms of their precision and speed. For instance, the relative precision of the UFIDA results in Table 2 was 2.4 to 4.8%, while the relative precision of the ultrafiltration results for the same samples was over two-fold larger, ranging from 8.1 to 9.7%. A similar trend was noted for the control serum results in Table 3. The worse precision of the ultrafiltration results is thought to be the combined result of 1) the multiple, manual steps that are involved in this method and 2) the random experimental variations that were noted in nonspecific binding of phenytoin to the ultrafiltration membrane. In contrast to this, the UFIDA method was performed as an automated system that did not require any sample pretreatment steps, which probably contributed to the better precision of this method versus that for ultrafiltration.

In terms of speed, UFIDA gave results for each sample within 2-3 min of injection, with a total run time of 20 min per cycle. For ultrafiltration, the minimum time required for one sample was approximately 1 h, which included 45 min to perform the ultrafiltration and 15 min to conduct the HPLC analysis of the filtrate's free phenytoin content. For UFIDA, a total of 15 h was required for a triplicate analysis of all the samples and standards used to generate the data in Table 2. Although ultrafiltration has a much higher analysis time per sample, up to 15 samples could be centrifuged simultaneously in this approach, giving it an overall sample throughput comparable to that of the UFIDA method (i.e., 14 h for analysis of all the samples and standards used in Table 2). However, it should be kept in mind that the throughput of the

UFIDA method could be increased by almost two-fold by using sequential injections of samples after each application of the labeled phenytoin, as demonstrated earlier in Figure 7B. This throughput could be improved even further through the use of multiple immunoextraction columns and a multiport valve for flow-splitting. Thus, UFIDA has the capability of not only providing a faster analysis per sample than ultrafiltration but also providing a higher sample throughput in some applications. Table 2. Determination of free phenytoin fractions in phenytoin/HSA mixtures by ultrafiltration or a UFIDA method^a

Measured free phenytoin cone. (μM) Total cone. Total cone.

HSA (μM) phenytoin (μM) Ultrafiltration UFIDA

550 30 4.9 (+0.4) 4.7 (+0.2)

550 35 5.7 (+0.5) 5.5 (+0.2)

550 40 6.6 (+0.6) 6.4 (± 0.2)

550 45 7.5 (+0.7) 7.2 (±0.2)

550 50 8.3 (+0.7) 8.1 (±0.2)

650 30 4.2 (±0.4) 4.1 (±0.1)

650 35 4.9 (±0.4) 4.8 (±0.1)

650 40 5.6 (±0.5) 5.5 (±0.1)

650 45 6.4 (±0.6) 6.2 (±0.2)

650 50 7.1 (±0.6) 6.9 (±0.2)

750 30 3.7 (±0.3) 3.5 (±0.1) 750 35 4.3 (+ 0.4) 4.2 (± 0.2)

750 40 4.9 (± 0.4) 4.8 (± 0.2)

750 45 5.6 (± 0.5) 5.4 (± 0.2)

750 50 6.2 (± 0.6) 6.1 (± 0.2)

^aAll measurements were performed at 37⁰C in pH 7.4, 0.067 M phosphate buffer. The values for ultrafiltration have been corrected for nonspecific binding to the membrane. The numbers in parentheses represent a range of ± 1 SD.

Table 3. Determination of free phenytoin fractions in serum samples by ultrafiltration or a UFIDA method^a

Measured free phenytoin cone. (μM)

Total cone, phenytoin (μM) Ultrafiltration UFIDA

10 1.32 (± 0.34) 1.27 (± 0.05)

20 2.81 (± 0.42) 2.70 (± 0.12)

40 6.11 (± 0.44) 5.99 (± 0.14)

^aAll measurements were performed in pH 7.4, 0.067 M potassium phosphate buffer and 37⁰C. The values for ultrafiltration have been corrected for nonspecific binding to the membrane. The values in parentheses represent a range of ± 1 SD. Non-Patent Publications

Various patent and non-patent publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

(1) Clarke, W.; Schiel, J. E.; Moser, A.; Hage, D. S. Anal. Chem. 2005, 77, 1859- 1866.

(2) Clarke, W.; Chowdhuri, A. R.; Hage, D. S. Anal. Chem. 2001, 73, 2157-2164.

(3) Lunde, P. K. M.; Rane, A.; Yaffe, S. J. Clin. Pharmacol. Ther. 1970, 11, 846.

(4) Barre, J.; Delion, F.; Tillement, J. P. Therapeut. Drug Monitor. 1988, 10, 133.

(5) Burt, M.; Anderson, D. C; Kloss, J.; Apple, F. S. Clin. Chem. 2000, 46, 1132- 1135.

(6) Bereczki, A.; Horvath, V. Anal. Chim. Acta 1999, 391, 9-17.

(7) Hara, S.; Hagiwara, F.; Ono, N.; Kuroda, T. Anal Sci. 1999, 15, 371-375.

(8) Chen, J.; Ohnmacht, C. M.; Hage, D. S. J. Chromatogr. B 2004, 809, 137-145.

(9) P. C. T. S. U. Drug Information for the Health Care Professional, 17 ed.; Rand McNaIIy: Tauton, 1997.

(10) Johansen, K.; Krogh, M.; Andresen, A. T.; Christophersen, A. S.; Lehne, G.; Rasmussen, K. E. J. Chromatogr. B 1995, 669, 281-288.

(11) Kodama, H.; Kodama, Y.; Shinozawa, S.; Kanemaru, R.; Todaka, K.; Mitsuyama, Y. J. Clin. Pharm. Therapeut. 1998, 23, 361-365. (12) Roberts, W. L.; Annesley, T. M.; De, B. K.; Moulton, L.; Juenke, J. M.; Moyer, T. Therapeut. Drug Monitor. 2001, 23, 148-154.

(13) Warner, A.; Privitera, M.; Bates, D. Clin. Chem. 1998, 44, 1085-1095.

(14) Gurley, B. J.; Marx, M.; Olsen, K. J. Chromatogr. B 1995, 670, 358-364.

(15) Troupin, A. S.; Shaw, L. M. Epilepsia 1985, 26, 455-459. (16) Jack, L.; Cunningham, C; Watson, I. D.; Stewart, M. Ann. Clin. Biochem.

1986, 23, 603-607. (17) Lu-Steffes, M.; Pittluck, G. W.; Jolley, M. E.; Panas, H. N.; Olive, D. L.;

Wang, C-H. J.; Nystrom, D. D.; Keegan, C. L.; David, T. P.; Stroupe, S. D.

Clin. Chem. 1982, 28, 2278-2282. (18) McGregor, A. R.; Crookall-Greening, J. O.; Landon, J.; Smith, D. S. Clin.

Chim. Acta 1978, 83, 161-166. (19) Melten, J. W.; Wittebrood, A. J.; Hubb, J. J.; Faber, G. H.; Werner, J.; Faber, D. B. J. Pharmaceut. Set 1985, 74, 692-694.

(20) Miller, T. D.; Pinkerton, T. C. Anal. Chim. Acta 1985, 170, 295-300.

(21) Oellerich, M.; Mueller-Vahl, H. Clin. Pharmacokin. 1984, 9, 61-70. (22) Popelka, S. R.; Miller, D. M.; Holen, J. T.; Kelso, D. M. Clin. Chem. 1981, 27, 1198-1201.

(23) Bertucci, C; Bartolini, M.; Andrisano, G. V. J. Chromatogr., B 2003, 797, 111.

(24) Rippel, G.; Corstjens, H.; Billiet, H. A.; Frank, J. Electrophoresis 1997, 18, 2175.

(25) Heegaard, N. H. H. J. MoI Recogn. 1998, 11, 141.

(26) Heegaard, N. H. H. Electrophoresis 2003, 24, 3879.

(27) Wang, H.; Zou, H.; Zhang, Y. Anal. Chem. 1998, 70, 373-377.

(28) Nelson, M. A.; Reiter, W. S.; Hage, D. S. Biomed. Chromatogr. 2003, 17, 188-200.

(29) Reng, X.; Draney, D. R. LabPlus Int. 2004, 18, 8.

(30) Sowell, J.; Parihar, R.; Patonay, G. J. Chromatogr., B 2001, 752, 1.

(31) Miki, S.; Kaneta, T.; Imasaka, T. J. Chromatogr., A 2005, 1066, 197.

(32) Castle, P. E.; Phillips, T. M.; Hildesheim, A.; Herrero, R.; Bratti, C. M.; Rodriguez, A. C; Morera, L. A.; Pfeiffer, R.; Hutchinson, M. L.; Pinto, L. A.;

Schiffman, M. J. Biochem. Biophys. Methods 2003, 12, 1449.

(33) Dodge, A.; Fluri, K.; Verpoorte, E.; de Rooij, N. F. Anal. Chem. 2001, 73,

(34) Phillips, T. M. J. Biochem. Biophys. Methods 2001, 49, 253.

(35) Ohnmacht, C. M.; Chen, S.; Tong, Z. J. Chromatogr. B 2006, 836, 83-91. (36) Clarke, W.; Beckwith, J. D.; Jackson, A.; Reynolds, B.; Karle, E. M.; Hage, D. S. J. Chromatogr. A 2000, 888, 13-22.

(37) Clarke, W.; Hage, D. S. Anal. Chem. 2001, 73, 1366-1373.

(38) Middendorf, L.; Amen, J.; Bruce, R.; Draney, D.; DeGraff, D.; Gewecke, J.; Grone, D.; Humphrey, P.; Little, G.; Lugade, A.; Narayanan, N.; Oommen, A.; Osterman, H.; Peterson, R.; Rada, J.; Raghavachari, R.; Roemer, S. In Near- infrared dyes for high technology applications; Daehne, S., Ed.; Kluwer Academic: Dordrecht, The Netherlands, 1998; p 21. (39) Kiec-Kononowicz, K.; Zejc, A.; Byrtus, H. Pol. J. Chem. 1984, 58, 585-591.

(40) Khan, M. N. J. Org. Chem. 1996, 61, 8063-8068.

(41) Lauer, R.; Zenchoff, G. J. Heterocyclic Chem. 1979, 16, 339-340.

(42) Shaffer, J. W.; Sheasley, R.; Winstead, M. B. J. Med. Chem. 1967, 10, 739. (43) Hermanson, G. T. Bioconjugate Techniques; Academic Press, Inc.: San Diego, 1996.

(44) Ruhn, P. F.; Garver, S.; Hage, D. S. J. Chromatogr. 1994, 669, 9.

(45) Chattopadhyay, A.; Hage, D. S. J. Chromatogr. 1997, 758, 255.

(46) Larsson, P. O. Methods Enzymol. 1984, 104, 2121. (47) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.;

Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 16.

(48) Hage, D. S. J. Chromatogr. B 2002, 768, 3-30.

(49) Argyle, C. J.; Kinniburgh, D. W.; Costa, R.; Jennison, T. Therapeut. Drug Monitor. 1984, 6, 117.

(50) Ohnmacht, C. M. Ph.D. Dissertation, University of Nebraska-Lincoln, Lincoln, 2006.

(51) Kim, H. S.; Hage, D. S. In Handbook of Affinity Chromatography, 2nd ed.; Hage, D. S., Ed.; CRC Press: Boca Raton, FL, 2005; Chapter 3. (52) Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand Techniques; Academic Press: New York, 1992.

(53) Hage, D. S.; Phillips, T. M. In Handbook of Affinity Chromatography, 2nd ed.; Hage, D. S., Ed.; CRC Press: Boca Raton, FL, 2005; Chapter 6.

(54) Van Regenmortel, M. H. V. Structure of Antigens, Vol. 1 ; CRC Press: Boca Raton, FL, 1992.

(55) Butler, J. E. Immunochemistry of Solid-Phase Immunoassay; CRC Press: Boca Raton, FL, 1991.

(56) Beck, D. E.; Farringer, J. A.; Ravis, W. R.; Robinson, C. A. Clin. Pharm. 1987, 6, 888-894. (57) Oates, M. R.; Clarke, W.; Zimlich, A. I.; Hage, D. S. Anal. Chim. Acta 2002,

470, 37-50. (58) Wiechmann, M. Hoppe-Seyler's Z. Physiol. Chem. 1977, 358, 967-980.

Claims

What is claimed is:

1. A method to determine the concentration of a free analyte fraction in at least one sample, the sample comprising a bound analyte fraction and the free analyte fraction, the free analyte fraction and the bound analyte fraction comprising free analyte and bound analyte, respectively, the method comprising:

(a) applying a labeled analyte, the labeled analyte being a labeled analog of the free analyte, in or approaching a saturating quantity to an affinity column having an active layer that binds at least some of the labeled analyte and selectively binds the free analyte relative to the bound analyte, wherein the active layer separates the free analyte fraction from the bound analyte fraction of the sample in the millisecond time domain;

(b) removing excess labeled analyte, if any, from the affinity column having the active layer with the bound labeled analyte;

(c) applying the sample to the affinity column, having the active layer with the bound labeled analyte and from which excess labeled analyte has been removed, thereby producing a displacement of the labeled analyte from the affinity column; (d) detecting a signal from the displacement of the labeled analyte from the affinity column by binding of the free analyte fraction of the sample to the affinity column; and

(e) determining from the signal the concentration of free analyte present in the sample.

2. The method of claim 1 wherein the active layer comprises support particles or support material derivatized with a binding agent.

3. The method of claim 2 wherein the active layer is from about 10 microns to about 1.1 millimeters in length.

4. The method of claim 2 wherein the active layer is at least about 60 microns in length.

5. The method of claim 2 wherein the binding agent is selected from the group consisting of antibodies, aptamers, antibody fragments, synthetic molecular imprints, antibody related molecules and recombinant proteins.

6. The method of claim 5 wherein the binding agent has a high binding affinity for the free analyte fraction.

7. The method of claim 6 wherein the binding agent is antibodies.

8. The method of claim 7 wherein the binding agent has a binding affinity for the free analyte fraction from about 10² to about 10⁶ M^'1.

9. The method of claim 7 wherein the binding agent has a binding affinity for the free analyte fraction greater than about 1O M^" .

10. The method of claim 7 wherein the free analyte fraction displaces the labeled analyte about 1 to about 500 milliseconds after injection of the sample into the affinity column.

11. The method of claim 10 wherein the free analyte fraction displaces the labeled analyte about 1 to about 100 milliseconds after injection of the sample into the affinity column.

12. The method of claim 10 wherein the signal is detected from the free analyte fraction by an off-line method or an on-line method.

13. The method of claim 12 wherein the signal is detected by an on-line method with direct detection.

14. The method of claim 12 wherein the signal is detected by near infrared fluorescent absorbance, immunoassay, mass spectrometry, gas chromatography, ultraviolet absorbance, fluorescence detectors, or electrochemical detectors.

15. The method of claim 14 wherein the label of the labeled analyte is one or more near infrared fluorescent cyanine dyes.

16. The method of claim 15 wherein the signal is detected by near infrared fluorescent absorbance using an excitation wavelength between about 670 and about 800 nm and an emission wavelength between about 670 and about 1000 nm.

17. The method of claim 1 wherein the sample comprises a biological fluid comprising the bound analyte fraction and the free analyte fraction.

18. The method of claim 17 wherein the signal is detected from the free analyte fraction by an off-line method or an on-line method.

19. The method of claim 18 wherein the biological fluid is selected from the group consisting of blood, plasma, urine, cerebrospinal fluid, a tissue sample, and intracellular fluid.

20. The method of claim 19 wherein the active layer has a number of active binding sites and has a binding capacity corresponding to a ratio of the number of active binding sites in moles to amount in moles of free analyte present in the sample between about 1 : 1 to about 10:1.

21. The method of claim 20 wherein the sample is injected onto the column at a flow rate of about 0.01 to about 9.0 milliliters per minute.

22. The method of claim 12 wherein the analyte is selected from the group consisting of a drug, hormone, peptide, enzyme inhibitor, toxin, metal ion, fatty acid, bilirubin or any other endogenous or exogenous compound

23. The method of claim 22 wherein the analyte is phenytoin.

24. The method of claim 23 wherein the binding agent of the active layer is anti- phenytoin antibody.

25. The method of claim 24 wherein the label of the labeled phenytoin is IRDye 800 CW dye (iV-hydroxysuccinimide, or NHS ester).

26. The method of claim 25 wherein the signal is detected by near infrared fluorescent absorbance using an excitation wavelength of 770 nm and an emission wavelength between about 780 and about 900 nm.

27. The method of claim 26 wherein the detection has a limit of about 500 pM to about 10 μM of free phenytoin.

28. A method to determine the concentration of a free analyte fraction in at least two samples, each sample comprising a bound analyte fraction and the free analyte fraction, the free analyte fraction and the bound analyte fraction comprising free analyte and bound analyte, respectively, the method comprising:

(c) applying the samples in sequence to the affinity column, having the active layer with the bound labeled analyte and from which excess labeled analyte has been removed, thereby producing a displacement of the labeled analyte from the affinity column; (d) detecting signals from the displacement of the labeled analyte from the affinity column by binding of the free analyte fraction of the sample to the affinity column in the sequence of their application; and

(e) determining the concentration of free analyte present in the samples from the signals corresponding to the sequence in which the samples were applied.

29. The method of claim 28 wherein the active layer comprises support particles or support material derivatized with a binding agent.

30. The method of claim 29 wherein the active layer is from about 10 microns to about 1.1 millimeters in length.

31. The method of claim 29 wherein the active layer is at least about 60 microns in length.

32. The method of claim 29 wherein the binding agent is selected from the group consisting of antibodies, aptamers, antibody fragments, synthetic molecular imprints, antibody related molecules and recombinant proteins.

33. The method of claim 32 wherein the binding agent has a high binding affinity for the free analyte fraction.

34. The method of claim 33 wherein the binding agent is antibodies.

35. The method of claim 34 wherein the binding agent has a binding affinity for the free analyte fraction from about 10² to about 10⁶ M^'1.

36. The method of claim 34 wherein the binding agent has a binding affinity for the free analyte fraction greater than about 10⁶ M^"1.

37. The method of claim 34 wherein the free analyte fraction displaces the labeled analyte about 1 to about 500 milliseconds after injection of the sample into the affinity column.

38. The method of claim 37 wherein the free analyte fraction displaces the labeled analyte about 1 to about 100 milliseconds after injection of the sample into the affinity column.

39. The method of claim 37 wherein the signal is detected from the free analyte fraction by an off-line method or an on-line method.

40. The method of claim 39 wherein the signal is detected by an on-line method with direct detection.

41. The method of claim 39 wherein the signal is detected by near infrared fluorescent absorbance, immunoassay, mass spectrometry, gas chromatography, ultraviolet absorbance, fluorescence detectors, or electrochemical detectors.

42. The method of claim 41 wherein the label of the labeled analyte is one or more near infrared fluorescent cyanine dyes.

43. The method of claim 42 wherein the signal is detected by near infrared fluorescent absorbance using an excitation wavelength between about 670 and about 800 nm and an emission wavelength between about 670 and about 1000 nm.

44. The method of claim 28 wherein the sample comprising a biological fluid comprising the bound analyte fraction and the free analyte fraction.

45. The method of claim 44 wherein the biological fluid is selected from the group consisting of blood, plasma, urine, cerebrospinal fluid, a tissue sample, and intracellular fluid.

46. The method of claim 45 wherein the active layer has a number of active binding sites and has a binding capacity corresponding to a ratio of the number of active binding sites in moles to amount in moles free analyte present in the sample between about 1:1 to about 10:1.

47. The method of claim 45 wherein the sample is injected onto the column at a flow rate of about 0.01 to about 9.0 milliliters per minute.

48. The method of claim 39 wherein the analyte is selected from the group consisting of a drug, hormone, peptide, enzyme inhibitor, toxin, metal ion, fatty acid, bilirubin or any other endogenous or exogenous compound

49. The method of claim 48 wherein the analyte is phenytoin.

50. The method of claim 49 wherein the binding agent of the active layer is anti- phenytoin antibody.

51. The method of claim 50 wherein the label of the labeled phenytoin is IRDye 800 CW dye (iV-hydroxysuccinimide, or NHS ester).

52. The method of claim 51 wherein the signal is detected by near infrared fluorescent absorbance using an excitation wavelength of 770 nm and an emission wavelength between about 780 and about 900nm.

53. The method of claim 52 wherein the detection has a limit of about 500 pM to about 10 μM of free phenytoin.