HPLC

October 28, 2009 — Tosoh’s advanced HPLC systems are fully automated systems that rapidly and precisely separate hemoglobins found naturally in human blood. Suitable for high throughput HbA1c testing in the laboratory or clinic environment in addition to rapid Hemoglobinopathy screening, Tosoh HPLC solutions provide a reliable, accurate and precise service at all times.

G8 HPLC Analyzer

G8 HPLC Analyzer

The key applications of their analysis modes are as follows:

- Follow-up of diabetic patients
HPLC is considered the “Gold Standard” technology in the follow-up of the plasma glucose concentration of diabetic patients over time, via the measurement of HbA1c (= glycated hemoglobin fraction). This technique was used in the ground breaking DCCT (Diabetes Control and Complications Trial) study.

To follow-up the plasma glucose concentrations over time the “HbA1c analysis mode” should be used. In this mode pre-determined windows are set in the software to detect the presence of Hemoglobins A1a, A1b, F, l-A1c, s-A1c and A0. Calibration against DCCT (NGSP) or IFCC allows the quantitation of the stabile A1c fraction, with other windows allowing presumptive identification of various variant haemoglobins.

- Screening for β-thalassemia and hemoglobinopathies
In the aid of diagnosing β-thalassemia and hemoglobinopathies the “β-thalassemia analysis mode” should be used. In this mode pre-determined windows are set in the software to detect the presence of Hemoglobins F, A0, A2, D+, S+ and C+. Calibration allows the quantitation of HbF and HbA2, with all other windows allowing the presumptive identification of various variant haemoglobins.

High Performance Liquid Chromatography
High performance liquid chromatography (HPLC) is the gold standard methodology for the HbA1c test. By manipulating the flow rate of the liquid (i.e. mobile phase) and the temperature of the HPLC column (i.e. stationary phase), it is possible to change the chromatographic display.

Early theories on chemical compounds were determined using simple methods (i.e. LC-MS and reverse-phase) and reagents (i.e. acetic acid and acetonitrile). However, since its humble beginning in research, HPLC is now widely used for diagnostics as the main technology in hospital departments. Furthermore, due to the numerous improvements in the performance of the columns and solvents, the chromatographic quality has become so accurate that a gradient can be used for customized elution patterns.

Tosoh G8 HPLC Analyzer
Tosoh designed the G8 from the user perspective, enhancing overall safety and convenience of operation. Ion-exchange HPLC is the gold standard for HbA1c diabetes measurement. Ion-exchange HPLC was used in the Diabetes Control and Complications Trial in the United States.

The Tosoh G8 HPLC Analyzer provides direct determination of stable HbA1c. The system is used for in vitro diagnostic measurement of HbA1c in blood specimens. The G8 uses a non-porous column and microcomputer technology to quickly and accurately measure the stable portion of HbA1c as a percentage of the total amount of hemoglobin present in the sample. The G8 provides accurate and precise separation of HbA1c from other hemoglobin fractions. Off-line pretreatment is not required, and there is no interference from the labile portion of HbA1c.

The analyzer dilutes the whole blood specimen with Hemolysis & Wash Solution and then injects a small volume of the treated specimen onto the TSKgel Glyco HSi Variant Column. Separation is achieved by utilizing differences in ionic interactions between the cation exchange group on the column resin surface and the hemoglobin components in a step gradient elution. The hemoglobin fractions (designated as A1a, A1b, F, LA1c , SA1c, A0, and H-V0, H-V1, H-V2) are subsequently removed from the column material by performing a step-wise elution using Elution Buffers HSi Variant 1, 2, and 3 that have specific salt and pH concentrations.

The separated hemoglobin components pass through the LED photometer flow cell where changes in absorbance are measured at 415nm. The G8 software integrates and reduces the raw data, and then calculates the relative percentages of each hemoglobin fraction. The print-out consists of the numerical results and the chromatogram. This represents the changes in absorbance versus retention time for each peak fraction. An analysis requires only 1.6 minutes.

HPLC principles and the G8 Analyzer