The launch of porous sub-2 μm particle stationary phases, combined with the simultaneous commercialization of compatible chromatographic instruments for these column geometries,^1,2 has increased the amount of liquid chromatography (LC) development, particularly in the field of high-throughput separations.

Because chromatographic efficiency and mobile phase flow-rate are inversely proportional to particle size, a significant decrease in analysis time can be achieved while maintaining or enhancing chromatographic performance.^3,4 However, one major constraint remains: the significant backpressure generated by small particles in optimal flow-rate conditions. This approach is known as UPLC (ultra performance liquid chromatography) or ultra high pressure liquid chromatography (UHPLC), according to the system provider. Numerous applications can be found in the literature with column lengths usually ranging from 50–150 mm^5–8 and, less frequently, with shorter column lengths.⁹

In the pharmaceutical industry, the quality control (QC) of a formulation remains relatively simple because the number of active compounds, excipients and impurities is generally limited. As mentioned in various Pharmacopoeia, the separation is often performed with a conventional HPLC column (i.e., 150–250 mm in length) packed with 5 μm particles.¹⁰ However, there is a need to both control a large number of samples every day and reduce the response time delivery. Therefore, the use of short columns packed with sub-2μm particles is of the utmost interest.

Experiment

Chemicals: Butylparaben was obtained from Sigma-Aldrich (Steinheim, Germany). Standards of bupivacaine HCl and atropine sulphate were kindly provided by Sintetica SA (Mendrisio, Switzerland), while clonidine was purchased from Fluka (Buchs, Switzerland). Degradation products, namely tropic acid and 2,6-dimethylaniline were both supplied by Fluka.

Injectable solution of clonidine 0.6 mg/mL was kindly provided by the "Pharmacie Interhospitalière de la Côtequot; (Morges, Switzerland). Bupivacaïne 0.5% and rapidocaïne 0.5% were obtained from Sintetica SA. Atropine 0.5 mg/mL was purchased from Amino AG (Neuenhof, Switzerland).

Acetonitrile was of HPLC gradient grade from Panreac Quimica (Barcelona, Spain). Water was obtained from a Milli-Q Waters Purification System from Millipore (Bedford, Massachusetts, USA). Phosphate buffers 50 mM at pH 7.0 and 7.4 were prepared with an adapted quantity of anhydrous di-potassium hydrogen phosphate and potassium dihydrogen phosphate from Fluka. Buffer solutions were prepared using the Phoebus software 1.0 from Analis (Namur, Belgium) and pH measured with a Metrohm pH meter (Herisau, Switzerland).

Equipment: Chromatographic experiments were performed on Waters Acquity UPLC system (Milford, Massachusetts, USA). This instrument included a binary solvent manager with a maximum flow-rate of 2 mL/min, an autosampler with an injection loop volume of 2 μL (1 μL was injected in partial loop mode), a UV/vis programmable detector, a column manager with an oven (set at 30 ºC). For all separations, the UV detector time constant was set at 25 ms and the data sampling rate at 80 Hz.