Minimizing Carry-over for High Throughput Analysis
A LC-MS system consisting of an Agilent 1260 Infinity HPLC and Agilent 6120 Quadrupol LC-MS equipped with a multimode ion source (set on ESI mode) was used. A PAL RTC sample preparation system with a LC/MS-Tool, high pressure injection valve (VICI C72VC-6676D-CTC) with a 2 µL loop was used. For separation a high throughput gradient HPLC method of 2 minutes cycle time was established using an Agilent Zorbax SB C18 Column (3 µm, 2.1x50mm) for chlorhexidine injections.
The PAL RTC offers five major wash tasks for carry-over minimization, such as post clean solvent 1 and 2, valve clean solvent 1 and 2, and stator wash. Solvent 2 is chosen according to analyte and matrix solvability, since it is used as the major wash step directly after each run. In contrast, solvent 1 is used to maintain reproducible conditions for subsequent analyses (therefore usually the eluent at starting conditions is used). The parameters of each wash step have been systematically screened using an on-column injection of chlorhexidine followed by fresh blanks. For chlorhexidine a mixture of water/methanol/acetonitrile/2-propanol containing 1% formic acid (for solvent 2) resulted in minimal carry-over.. A total of 5 strokes and 200 µL solvent 2 and two strokes and 90 µL solvent 1 for post clean and valve clean have been used respectively. Usinging this conditions a carryover of less than 20 ppm has been observed usuing wash cycles of less than 2 minutes for chlorhexidin. An optimized auto sampler wash protocol was developed and general guidelines for method development for HT LC-MS applications were established. The coloumn has been identified as the main source of carry-over.