Introduction

High speed HPLC separations have become increasingly important in a variety of different fields including pharmacology, food science, and agrochemistry. In general, conventional HPLC separations have been performed on columns packed with a stationary phase composed of 5 µm particles. By contrast, UHPLC system uses shorter columns packed with smaller sub-2 µm particles. This article details the analysis of a tuberculosis treatment composed of three compounds (rifampin ,isoniazid, and pyrazinamide) with a method outlined by USP to compare the performance of UHPLC with conventional HPLC.

LC-4000 UHPLC system

Experimental

The conventional and rapid sample analyses described below were made using an integrated UHPLC system configured for binary gradient with UV-visible detection.

Chromatographic conditions

Mobile Phase A: 0.01 M Na₂HPO₄/CH₃CN (96/4) (pH 6.8)
Mobile Phase B: 0.01 M Na₂HPO₄/CH₃CN (45/55) (pH 6.8)
Wavelength 238 nm
Sample 0.16 mg/L Rifampin, 0.08 mg/L Isoniazid, 0.43 mg/L Pyrazinamide, (in 0.01M Na₂HPO₄/CH₃OH (96/4))

Results

Figure 1 shows chromatogram of medicines using conventional HPLC. A flow rate of 1.5 mL/min was used with a CrestPak C18S column, 5 µm particle size (4.6 mm i.d. X 150 mm L). The analysis time was 9 min.

Figure 1 Chromatogram of medicines using column (4.6 mm I.D. x 150 mm) packed with 5 µm particles in diameter (1: Rifampin, 2: Isoniazid, 3: Pyrazinamide)

Figure 2 shows chromatogram of medicines using UHPLC. A flow rate of 0.8 mL/min, nearly half that of the HPLC separation, was used for elution of the sample using an X-PressPak C18S column with 2 µm particles (2.1 mm i.d. X 50 mm L). This resulted in an analysis time of 1.3 min, approximately 7 times faster than the HPLC separation. The gradient elution program of 1.5 min is 10 times shorter than that for HPLC.

Figure 2 Chromatogram of medicines using column (2.1 mm I.D. x 50 mm L.) packed with 2 µm particles in diameter (1: Rifampin, 2: Isoniazid, 3: Pyraziamide)

Table 1. Comparison between UHPLC and conventional HPLC.

Conclusion

We have demonstrated that UHPLC system offers rapid separations, up to 10 times faster than that of conventional HPLC, with improved sensitivity and efficiency whilst reducing mobile phase solvent use. This more efficient technique is also completely scaleable from traditional HPLC methods providing an attractive option for a wide range of samples.