Method of analyzing hemoglobins

Abstract

An object of the present invention is to provide a method for analyzing hemoglobins which can accurately separate hemoglobins in a short time by liquid chromatography.The present invention provides a method for analyzing hemoglobins by liquid chromatography which includes pre-treating a sample with an oxidant and a binder for trivalent heme iron.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for analyzing hemoglobins by separating hemoglobins by liquid chromatography.BACKGROUND ART

[0002] High-performance liquid chromatography (HPLC) analysis of hemoglobins is a widely used technique. Specifically, this technique is used, for example, to quantify a glycohemoglobin known as hemoglobin A1c or to analyze abnormal hemoglobins for diagnosis of diabetes. For example, a method utilizing liquid chromatography has been known which separates hemoglobin components in a diluted hemolyzed blood sample by a cation-exchange method based on the difference in positive charge among the hemoglobin components. A recent increase in patients with diabetes has also increased the number of cases requiring measurement of hemoglobin A1c. This tendency has created a demand for more accurate, less time-consuming measurement by HPLC.

[0003] Hemoglobins are present in the body in the forms of oxyhemoglobin that contains bound oxygen, deoxyhe...

Examples

example 1

[0075]A sample was prepared by diluting a hemoglobin S-containing blood sample 100-fold with a sample pre-treatment solution (phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100).

[0076]The used separation column was a column containing cation-exchange resin filler particles having sulfonic acid groups on the surfaces.

[0077]The used HPLC instrument was provided with an autosampler SIL-20AC (Shimadzu Corp.), a delivery pump LC-20AD (Shimadzu Corp.), a degasser DGU-20A5 (Shimadzu Corp.), a column oven CTO-20AC (Shimadzu Corp.), and a detector SPD-M20A (Shimadzu Corp.). The instrument was run under the following conditions:

[0078]eluent flow rate: 1.7 mL / min;

[0079]detection wavelength: 415 nm; and

[0080]amount of introduced sample: 10 μL.

[0081]Each portion of the sample was eluted and measured using the following eluents for the respective periods of time:

[0082]until 0.5 minutes after the start: eluent 1 (40 mmol / L phosphate buffer (pH 5.4) containing 60 mmol / L sodium perchlo...

example 2

[0090]A sample was prepared by dissolving glycohemoglobin control level II (Sysmex Corp.) in water for injection (200 μL), and further diluting the solution 100-fold with a sample pre-treatment solution (10 mmol / L phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100).

[0091]Another sample was prepared by diluting a hemoglobin S-containing blood sample 100-fold with the sample pre-treatment solution (10 mmol / L phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100).

[0092]The same separation column as that of Example 1 was used.

[0093]The same HPLC instrument as that of Example 1 was run under the following conditions:

[0094]flow rate: 1.7 mL / min;

[0095]detection wavelength: 415 nm; and

[0096]amount of introduced sample: 10 μL.

[0097]Each sample was eluted and measured by linear gradient of two eluents:

[0098]first eluent: eluent 6 (20 mmol / L phosphate buffer (pH 5.4) containing 30 mmol / L sodium perchlorate, 1 mmol / L sodium nitrite, and 1 mmol / L sodium azide); and

[0099]...

example 3

[0101]A sample was prepared by dissolving glycohemoglobin control level II (Sysmex Corp.) in water for injection (200 μL), and further diluting the solution 100-fold with a sample pre-treatment solution (10 mmol / L phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100, 1 mmol / L sodium nitrite, and 1 mmol / L sodium azide).

[0102]Another sample was prepared by diluting a hemoglobin S-containing blood sample 100-fold with the sample pre-treatment solution (10 mmol / L phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100, 1 mmol / L sodium nitrite, and 1 mmol / L sodium azide).

[0103]The samples were measured in the same manner as in Example 2, except that eluent 3 (40 mmol / L phosphate buffer (pH 8.0) containing 0.8% by weight Triton X-100, 300 mmol / L sodium perchlorate, and 1 mmol / L sodium azide) used in Example 1 was used as the second eluent. FIG. 4 are the resulting chromatograms.

TECHNICAL FIELD

[0001] The present invention relates to a method for analyzing hemoglobins by separating hemoglobins by liquid chromatography.BACKGROUND ART

[0002] High-performance liquid chromatography (HPLC) analysis of hemoglobins is a widely used technique. Specifically, this technique is used, for example, to quantify a glycohemoglobin known as hemoglobin A1c or to analyze abnormal hemoglobins for diagnosis of diabetes. For example, a method utilizing liquid chromatography has been known which separates hemoglobin components in a diluted hemolyzed blood sample by a cation-exchange method based on the difference in positive charge among the hemoglobin components. A recent increase in patients with diabetes has also increased the number of cases requiring measurement of hemoglobin A1c. This tendency has created a demand for more accurate, less time-consuming measurement by HPLC.

[0003] Hemoglobins are present in the body in the forms of oxyhemoglobin that contains bound oxygen, deoxyhe...

Examples

example 1

[0075]A sample was prepared by diluting a hemoglobin S-containing blood sample 100-fold with a sample pre-treatment solution (phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100).

[0076]The used separation column was a column containing cation-exchange resin filler particles having sulfonic acid groups on the surfaces.

[0077]The used HPLC instrument was provided with an autosampler SIL-20AC (Shimadzu Corp.), a delivery pump LC-20AD (Shimadzu Corp.), a degasser DGU-20A5 (Shimadzu Corp.), a column oven CTO-20AC (Shimadzu Corp.), and a detector SPD-M20A (Shimadzu Corp.). The instrument was run under the following conditions:

[0078]eluent flow rate: 1.7 mL / min;

[0079]detection wavelength: 415 nm; and

[0080]amount of introduced sample: 10 μL.

[0081]Each portion of the sample was eluted and measured using the following eluents for the respective periods of time:

[0082]until 0.5 minutes after the start: eluent 1 (40 mmol / L phosphate buffer (pH 5.4) containing 60 mmol / L sodium perchlo...

example 2

[0090]A sample was prepared by dissolving glycohemoglobin control level II (Sysmex Corp.) in water for injection (200 μL), and further diluting the solution 100-fold with a sample pre-treatment solution (10 mmol / L phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100).

[0091]Another sample was prepared by diluting a hemoglobin S-containing blood sample 100-fold with the sample pre-treatment solution (10 mmol / L phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100).

[0092]The same separation column as that of Example 1 was used.

[0093]The same HPLC instrument as that of Example 1 was run under the following conditions:

[0094]flow rate: 1.7 mL / min;

[0095]detection wavelength: 415 nm; and

[0096]amount of introduced sample: 10 μL.

[0097]Each sample was eluted and measured by linear gradient of two eluents:

[0098]first eluent: eluent 6 (20 mmol / L phosphate buffer (pH 5.4) containing 30 mmol / L sodium perchlorate, 1 mmol / L sodium nitrite, and 1 mmol / L sodium azide); and

[0099]...

example 3

[0101]A sample was prepared by dissolving glycohemoglobin control level II (Sysmex Corp.) in water for injection (200 μL), and further diluting the solution 100-fold with a sample pre-treatment solution (10 mmol / L phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100, 1 mmol / L sodium nitrite, and 1 mmol / L sodium azide).

[0102]Another sample was prepared by diluting a hemoglobin S-containing blood sample 100-fold with the sample pre-treatment solution (10 mmol / L phosphate buffer (pH 7.0) containing 0.1% by weight Triton X-100, 1 mmol / L sodium nitrite, and 1 mmol / L sodium azide).

[0103]The samples were measured in the same manner as in Example 2, except that eluent 3 (40 mmol / L phosphate buffer (pH 8.0) containing 0.8% by weight Triton X-100, 300 mmol / L sodium perchlorate, and 1 mmol / L sodium azide) used in Example 1 was used as the second eluent. FIG. 4 are the resulting chromatograms.

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