On the extra-column band-broadening contributions of modern, very high pressure liquid chromatographs using 2.1 mm I.D. columns packed with sub-2 μm particles

The efficiencies of two narrow bore columns (100 mm and 50 mm × 2.1 mm) packed with 1.7 μm totally porous BEH-C₁₈ particles were measured on two very high pressure liquid chromatographs (Acquity from Waters and 1290 Infinity HPLC System from Agilent) operating at maximum pressures of 1034 and 1200 bar, respectively. The probe compounds were a mixture of uracil, acetophenone, toluene, and naphthalene eluted in a 50/50 (v/v) solution of acetonitrile and water at 303 K with a flow rate of 0.40 mL/min. The apparent efficiencies of columns, which lumps the consequences of band broadening due to the column and the system contributions, may depend much on the extra-column volumes of the instruments used. Actually, it is known for a long time that the apparent column performance is strongly affected by the instrument characteristics, including the diameter of the connecting tubes, the injection technique (with or without needle seat capillary), and the detection cell volume. When the 1290 Infinity HPLC System is equipped with a needle seat, an inlet and an outlet connecting capillary tube with inner diameters around 115 μm, its extra-column variance for a 0.1 μL injection volume is 9.2 μL² while that of the Acquity instrument is 6.9 μL². Minor modifications suggested by their respective manufacturers allowed significant reductions of these variances, to 6.2 and 3.9 μL², respectively. Yet, in their optimized configurations and for weakly retained compounds (k ? 1), these modern, sophisticated instruments cannot provide more than 75% (1290 Infinity) and 85% (Acquity) of the maximum efficiency of a 2.1 mm × 50 mm BEH column. For more strongly retained compounds (k > 4), in contrast, they are both able to provide more than 95% of the maximum expected efficiency.     

Rapid ultraperformance liquid chromatography–tandem mass spectrometry method for quantification of oxcarbazepine and its metabolite in human plasma

A rapid and sensitive ultraperformance liquid chromatography tandem mass spectrometry assay was developed for the simultaneous analysis of oxcarbazepine and its main metabolite in human plasma. The assay involves a simple solid‐phase extraction procedure of 0.3 mL of human plasma and analysis was performed on a triple‐quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionization. Separation was achieved on an Acquity UPLC™ BEH C₁₈ column (50 × 2.1 mm, i.d., 1.7 µm) with isocratic elution at a flow‐rate of 0.25 mL/min and imipramine was used as the internal standard. The standard calibration curve was linear over the range 9.580–5070.205 ng/mL for oxcarbazepine (OXC) and 19.444–10290.800 ng/mL for 10,11‐dihydro‐10‐hydroxycarbamazepine (MHD), expressed by the linear correlation coefficient r², which was better than 0.995 for OXC and MHD. The intra‐ and inter‐day precision and accuracy of the quality control samples were within 10.0%. The recoveries were 81.0, 89.6 and 66.6% for OXC, MHD and imipramine, respectively. The total run time was 1.5 min only for each sample, which makes it possible to analyze more than 350 samples per day. Copyright © 2010 John Wiley & Sons, Ltd.     

Achieving the full performance of highly efficient columns by optimizing conventional benchmark high-performance liquid chromatography instruments

A series of experiments and measurements demonstrate the importance of minimizing the extra-column band broadening contribution of the instrument used. The combination of several measures allowed the achievement of the full potential efficiency of three Kinetex-C₁₈ columns, using a conventional liquid chromatograph. The first measure consists in minimizing the extra-column volume of the instrument, without increasing much its back pressure contribution, by changing the needle seat volume, the inner diameter and length of the capillary connectors, and the volume of the detector cell of a standard instrument (Agilent 1100). The second measure consists in injecting a volume of weak eluent (less than half the elution strength of the mobile phase) right after the sample, before the sample had time to reach the column. Experimental results show that these changes could provide most of the resolution expected from the true column performance. After the changes were made, the resolutions of the 2.1 mm ×$\times$ 50 mm, 4.6 mm ×$\times$ 50 mm, and 4.6 mm ×$\times$ 100 mm Kinetex-C₁₈ columns for compounds having retention factors close to 1 were increased by about 180, 35, and 30%, respectively. The resolutions obtained are then similar to those measured with advanced instruments like the Agilent 1200, the Agilent 1290 Infinity HPLC, and the Acquity chromatographs.     

Advantages of application of UPLC in pharmaceutical analysis

Ultra Performance Liquid Chromatography (UPLC) is a relatively new technique giving new possibilities in liquid chromatography, especially concerning decrease of time and solvent consumption. UPLC chromatographic system is designed in a special way to withstand high system back-pressures. Special analytical columns UPLC Acquity UPLC BEH C₁₈ packed with 1.7 μm particles are used in connection with this system.The quality control analyses of four pharmaceutical formulations were transferred from HPLC to UPLC system. The results are compared for Triamcinolon cream containing trimacinolone acetonide, methylparaben, propylparaben and triamcinolone as degradation product, for Hydrocortison cream (hydrocortisone acetate, methylparaben, propylparaben and hydrocortisone degradation product), for Indomethacin gel (indomethacin and its degradation products 4-chlorobenzoic acid and 5-methoxy-2-methylindoleacetic acid) and for Estrogel gel (estradiol, methylparaben, propylparaben and estrone as degradation product).The UPLC system allows shortening analysis time up to nine times comparing to the conventional system using 5 μm particle packed analytical columns. In comparison with 3 μm particle packed analytical columns analysis should be shortened about three times. The negative effect of particle decrease is back-pressure increase about nine times (versus 5 μm) or three times (versus 3 μm), respectively. The separation on UPLC is performed under very high pressures (up to 100 MPa is possible in UPLC system), but it has no negative influence on analytical column or other components of chromatographic system. Separation efficiency remains maintained or is even improved. Differences and SST parameters, advantages and disadvantages of UPLC are discussed.