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Field of the Invention
The present invention is broadly concerned with an improved, high speed HPLC assay for the determination of glycated hemoglobin in blood-derived analytes. More particularly, the invention pertains to a continuous assay method of this type wherein individual samples are continuously and sequentially analyzed with an injection-to-injection time between samples of less than 2 minutes.

Description of the Prior Art
PCT Publication WO 90/06516 describes a continuous HPLC system and method for the analysis of plural liquid proteinaceous samples, and particularly blood-derived samples, in order to accurately quantitate glycated and non-glycated blood fractions such as hemoglobins. In this system, the known technique of boronate affinity chromatography is used which relies on the fact that boronates will reversibly bind under appropriate conditions with the functional groups in glycated proteins. The methods described in this PCT Publication represent a significant breakthrough in the art and allow the user to assay, on an essentially automatic basis, a large number of individual analyte samples. The injection-to-injection times described in this publication are well above 2 minutes and up to about 8 minutes.
The Primus Corporation of Kansas City, Missouri has heretofore commercial-ized the assays described in the PCT Publication using elevated temperatures of up to

40 °C for plasma proteins and up to 50 °C for hemoglobin proteins. The minimum injection-to-injection time achieved with such methods has been 4 minutes for plasma proteins and 2 minutes for hemoglobin proteins. However, these injection-to-injection times are still deemed inadequate by many users, and therefore there is a need in the art for still further improvements serving to lessen the injection-to-injection times and thereby to increase productivity.

The present invention overcomes the problems outlined above and provides an improved HPLC assay for the determination of at least the glycated hemoglobin fraction present in blood-derived analyte samples. The invention represents a significant improvement over the assay described in PCT Publication WO 90/06516 which is incorporated by reference herein.
It has been found that by appropriate control of the HPLC column temperature and eluant flow rates, the time between successive analyses of individual samples

(normally termed the injection-to-injection time) can be reduced to less than 2 minutes. Generally speaking, in the method of the invention a sample containing glycated and non-glycated hemoglobin species is introduced into the HPLC column (normally polymer filled with m-aminophenylboronic acid (PBA) supported on the polymer fill), and the non-glycated and glycated fractions are separated using suitable mobile phases.

An analyses is then conducted of at least the glycated fraction, and usually both fractions, to give an accurate quantitative readout. In the assay, the HPLC column temperature is maintained at a level above 50 °C and up to about 60 °C (more preferably from above 50 ° C to about 55 ° C). Flow rates for the mobile phases passing through the column are from about 1-4 ml/min. and more preferably from about 1-3 ml/min.

Column pressures are dependent variables resulting from factors such as column packing particle sizes, flow rates and temperatures. Typically, levels of from about 300-1500 psi are observed. Sample sizes applied to the column normally range from about 3-38 micrograms of blood-derived analyte containing the glycated and non-glycated hemoglobin fractions, more preferably from about 3-15 micrograms.
In preferred practice, two mobile phase solutions are employed during each individual assay. These are generally referred to as mobile phases A and B. Mobile phase A is designed to equilibrate the HPLC column, create appropriate conditions for binding of the glycated hemoglobin fraction onto the column, and to transport the non-glycated fraction from the column. Mobile phase B on the other hand is designed to elute the bound glycated fraction, and for this purpose typically includes a polyol such as mannitol. The respective mobile phases can be used on a gradient basis, i.e., a linear or non-linear transition between the phases using phase A and phase B mixtures. Alternately, the phases may be abruptly changed without a mixed phase gradient.
By conducting the assay at the required elevated temperatures and mobile phase flow rates, the injection-to-injection time for successive samples can be reduced below 2 minutes, preferably from about 1.9 minutes and most preferably from about 1.4-1.75 minutes. This represents a decided advantage to a commercial laboratory, inasmuch as the number of samples analyzed per hour is significantly increased.

Figure 1 is a schematic representation of the preferred apparatus used in performing the HPLC assays of the invention;
Fig. 2 is a chromatogram produced by the preferred HPLC equipment as described in Example 1 ;
Fig. 3 is a chromatogram produced by the preferred HPLC equipment as described in Example 2; and
Fig. 4 is a chromatogram produced by the preferred HPLC equipment as described in Example 3.

Turning now to Fig. 1, an automated chromatographic device 10 useful in the invention is schematically illustrated. The device 10 includes a chromatographic column 12 having an input line 14 coupled to one end thereof, with an output line 16 connected to the opposing end. The preferred column is in the form of a tube filled with polymer particles of approximately 3-20 microns in diameter having pores of from about 300-1,000 A in diameter. PBA is bonded to the polymeric support by covalent coupling. The presently preferred column is distributed by the TosoHaas Company of Philadelphia, PA under the designation "Boronate-5PW." A sample injection system 18 is also provided including an autosampler 20 and injector 22, interconnected by means of line 24. The injector is in turn connected to input line 14 of column 12, via line 26.
The device 10 further includes a total of two reservoirs 28 and 30, respectively adapted for holding the mobile phases A and B. Two valves 34 and 36 are respectively coupled to an associated reservoir 28 and 30, with an output line 40 or 42 leading from a respective valve to a common output line 46. The line 46 is in turn connected with input line 14 of column 12, and has a pump 47 interposed therein. Alternately, use can be made of a single switching valve in place of the valves 34 and 36.
The device 10 also includes a spectrophotometric detector 48 of conventional design which is connected with output line 16 from column 12. The detector 48 is in turn electrically coupled with an integrating computer 50 which is connected to and controls a chart printout device 52. Electrical leads schematically illustrated as at 54, 55 and 56 respectively couple computer 50 with the autosampler 20 and injector 22, with the operations (not shown) of the valves 34-38, and with the motor (not shown) of pump 47. This allows complete control of the injection system 18 and the reservoir pump via computer 50.
The device 10 is preferably a commercially available Hewlett Packard Model HP 1090 automated chromatograph. The device 10 is provided with software for the control of the computer 50, the latter being described in detail in PCT Publication WO

As is apparent from the foregoing discussion, a sample 58 is injected via system 18, and the entire device 10 is then controlled by way of computer 50 to perform the entire analysis on an automated basis. The computer 50 together with the printout device 52 correspondingly develops a chromatogram, and measures the area under the analyte peaks, thereby providing an accurate protein analysis.
Preparation of sample 58 is essentially conventional. In the case of a blood sample, venous blood (typically 3-10 ml) collected via venipuncture or by fingerstick (50-100 μl). Freshly collected blood is mixed with sufficient anticoagulant, such as EDTA or heparin, to prevent clotting.
Generally speaking, two preferred variations of sample preparation are used for the glycated hemoglobin test. Both involve the preparation of a "hemolysate" or solution of blood in which the RBC cell membranes have been lysed or ruptured. In the first variation, whole blood is diluted 1 :100 in purified water or other lysing agent, which ruptures the RBC membranes. In the second variation, the blood sample is centrifuged, which separates the plasma from the cells. The plasma is removed and the hemolysate is prepared from the packed RBCs by diluting them 1 :200 in purified water or other lysing agents.
Hemolysates are transferred to glass or plastic sample vials, which are specially designed to fit the autosampler 20. An optional metal cap with a flexible septum is attached by means of a crimping tool to cover the top of the vial.
The most preferred mobile phase A designed for introduction into reservoir 28 is an aqueous solution containing 0.25 M ammonium acetate, 0.5 M sodium chloride, 5% ethyl alcohol, and sufficient sodium hydroxide to bring the pH of the solution to about 9. An antimicrobial preservative can also be added if desired. Mobile phase B is likewise an aqueous solution and includes 0.1 M mannitol, 0.15 M sodium chloride, and 5% ethyl alcohol; an antimicrobial preservative can also be added. As described above, various changes can be made in both the proportions and specific components of these mobile phases, the important factors being that the solutions be capable of separating the glycated and non-glycated fractions in the chromatograph.

Referring again to Fig. 1, computer 50 typically includes an output printer 52 coupled therewith, a data entry keyboard, and a floppy disk drive for receiving a memory device such as a floppy disk and for electronically retrieving instructions stored thereon. As those skilled in the art will appreciate, the memory device can also include a so-called "hard disk," compact optical disk, magnetic tape or any type of device on which electronically retrievable instructions can be stored.
In the preferred embodiment, more than one analytical method for operating liquid chromatograph 10 may be stored on the floppy disk. The utility of the preferred embodiment is not so limited, however, but rather is enhanced by the ability to include instructions for virtually any analytical method for operating the liquid chromatograph.

As an alternative, a separate memory device may be provided for each analytical method which can advantageously form a library of complete methods for operating liquid chromatograph 10.
In practice, the operator first ensures that the liquid samples to be analyzed are placed into autosampler 20, that reservoirs 28-30 contain the proper mobile phases, and that the proper column 12, specific to the analyses to be performed, is in place. The operator then places the selected disk in the disk drive and boots up computer 50. For purposes of analyzing samples containing glycated and non-glycated hemoglobin fractions, software described in the aforementioned PCT Publication is entirely suitable, although other options exist.
The following examples set forth preferred techniques in accordance with the invention. It is to be understood however, that these examples are presented by way of illustration only and nothing therein should be taken as a limitation upon the overall scope of the invention.

Example 1
In this example, a number of blood samples were assayed for glycated hemoglobin using the Hewlett Packard HP 1090 HPLC instrument and mobile phases A and B described above. In each instance, one part whole blood was diluted in 100 parts water and a sample injection volume of 5 μL of the diluted blood was used. The temperature of the HPLC column was maintained at 55 °C and a mobile phase flow rate was 2.4 ml/min.
The HPLC column was first equilibrated over 1.44 min. with 100% mobile phase A. The non-linear mobile phase gradient was carried out as follows. At the time of sample injection (i.e., time zero), the mobile phase consisted of 100% mobile phase B. This mobile phase condition was maintained for 0.3 min. From time 0.3 min until 0.31 min., the mobile phase was changed in a linear fashion to 100% mobile phase A. The mobile phase condition was maintained until the next sample injection (1.44 min. later). Thus, a total time of 1.75 min. elapsed from injection-to-injection.
In the described HPLC system, the sample is injected at a point in the flow path that is closer to the column than is the valve controlling the choice of mobile phases. As a result, there is a lag period between the time the sample reaches the column and the time of actual change in mobile phase composition in the column (such lag being determined by eluant flow rate and system geometry). Thus, while the valve controlling the selection of mobile phases is changed to deliver mobile phase B at the time of sample injection (time zero), the mobile phase B does not actually arrive at the column until a certain time after the valve changeover. Likewise, when the valve is again changed to mobile phase A, a similar time lag obtains. The following table sets forth the relevant times, valve positions, mobile phases in the column, analyte conditions in the column and detector conditions during an exemplary sample injection.

An exemplary chromatogram generated in this series of tests is illustrated in Fig. 2, where it will be seen that a clear separation was established between the glycated and non-glycated hemoglobin fractions. The total glycated hemoglobin according to the assay was 9.5%, the correct value.

Example 2
This example also involved the analysis of a series of blood samples using the equipment, mobile phases and sample preparation described in Example 1. In this instance, however, an HPLC column temperature of 60 °C was maintained with a mobile phase flow rate of 3.0 ml/min.
In particular, the HPLC column was equilibrated over 1.17 min. with 100%) mobile phase A. The non-linear mobile phase gradient included the following steps.

At the time of sample injection (time zero), the mobile phase consisted of 100% mobile phase B. This mobile phase condition was maintained for 0.25 min. From time 0.25 min. until time 0.26 min., the mobile phase was linearly changed to consist of 100% mobile phase A. This mobile phase condition was maintained until the next sample injection (1.17 min. later). Thus, a total time of 1.43 min. elapsed from injection-to-injection.
A sample chromatogram derived from this series of runs is provided as Fig. 3, and shows a clean separation between the glycated and non-glycated fractions.

Example 3
In this example, modular HPLC equipment obtained from Gilson Medical Electronics, Inc. of Middletown, WI was employed in lieu of the Hewlett Packard equipment. In particular, a Gilson Model 234 autoinjector with a Model 7010 Rheodyne valve was used, the latter having a 5 μL injection loop. In addition, Models 306 and 307 pumps were employed together with a Model 118 UV/VIS detector equipped with a 413 nm filter. The HPLC column was maintained at temperature with a custom-made heater similar to many commercially available heaters. The computer equipment was standard IBM compatible hardware.
In the assay, sample preparation was identical to that of Examples 1 and 2 using an injection volume of 5 μL and mobile phases A and B. Column temperature was maintained at 55 °C and mobile phase rate was maintained at 2.0 ml min. The column was first equilibrated over a 1.2 min. period with 100% mobile phase A. At the time of sample injection (time zero), the mobile phase consisted of 100% mobile phase B. This mobile phase condition was maintained for 0.3 min. At time 0.3 min., the mobile phase condition was changed abruptly to 100% mobile phase A. This mobile phase condition was maintained until the next injection (1.2 min. later). Thus, a total time of 1.5 min. elapsed from injection-to-injection.
Fig. 4 is a sample chromatogram derived from this series of runs.