Phase ratio column diameter reduction

The mono-, di-, and tri-phosphorylates of d4T were analysed by ion-pair LC-MS after lysis of the PBMC cell in Tris/methanol and centrifugation [50]. The supernatant was injected into the LC system with a 150x2.1-mm-ID Cjg column (5 pm) and a mobile-phase gradient of 70 to 35% solvent A (10 mmol/1 DMHA and 3 mmol/1 ammonium formate adjusted at pH 11.5) in solvent B (50% acetonitrile in 20 mmol/1 DMHA and 6 mmol/1 ammonium formate). Negative-ion ESI-MS was performed in SRM mode. The method enabled the direct measurement of the chain terminator ratio (d4T-triphosphate/deoxythymidine-triphosphate). Subsequently, the same group [51] reported modifications of this method, including simplifications of the sample pretreatment, replacement of the LC column for another type, and reduction of the column inner diameter from 2 mm ID to 0.32 mm ID. This improved method was applied to the determination of the phosphorylates of d4T, 3TC, and ddl. The sample throughput is 200 samples per week. The determination of intracellular AZT-triphosphate in PBMC [52], and the validation of the method for the determination of the ddl and d4T triphosphates was reported separately [53]. 

The trend of the liquid phase to spread on the column wall increase with the reduction of the ratio between the diameters of foe column and foe packing. (This b due to hi er value of > at higher packing diameters). 

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