18


September 2009


salt type


some impact on retention and peak shape, little impact on selectivity


salt concentration major impact on retention and peak shape, little impact on selectivity acid/base ratio


some impact on retention,moderate impact on selectivity


stationary phase major impact on selectivity and retention (need for screening still exists) Table 3: Summary of results from the LC-MS study of metaprolol


shown in Figure 4. This compares the separation of fluoxetine enantiomers on a 25 cm x 4.6 mm I.D. column to a 10 cm x 2.1


mm I.D. column, both packed with 5 µm Chirobiotic V2. The flow rate was adjusted to obtain the same linear velocity on both columns. By decreasing the flow rate on the short column, compared to the equivalent linear velocity on the larger column, equivalent resolution with dramatically improved sensitivity was obtained. The


CONDITIONS column:


Astec CHIROBIOTIC V2, 25 cm x 4.6 mm (black) or 10 cm x 2.1 mm I.D. (red), 5 μm particles


mobile phase: 10 mM ammonium formate in 10:90 v/v water:methanol


flow rate: temp.:


detector.: injection: sample:


1.0 (black), 0.1 (red) mL/min.


ambient


+ESI, m/z 310 2 μL


5 μg/mL in methanol Figure 4. Relative LC-MS response of fluoxetine enantiomers on CHIROBIOTIC V2 in reversed Phase


acetic acid/triethylamine in thismobile phase. In the investigation of differences between salt types, no selectivity was observed using one salt that was not observed when using the other two salts in any of the probemix separations, so choosing one of these as a generic screen would also be acceptable. A comparison of the results for various concentrations of ammoniumformate in methanol formetoprolol confirmed that retention of this base increases with decreasing concentration, indicating a dominating ionic interaction. Lowering salt concentration appears to assist selectivity. The results are summarized in Table 3. Figure 3 shows the results fromone such batch screen using a Chirobiotic TAG column in the polar ionicmode.


Flow rate studies An interesting characteristic of the Chirobiotic CSPs is that optimum flow rates for maximum efficiency are quite low. Using van Deemter curves (Figure 3), the optimum flow rates for analytical (25cm x 4.6 mm) columns are observed to be as low as 0.2 mL/min, with an almost 100% increase in efficiency over that at 1.0 ml/min. Results from method development screening run at 1.0 mL/min can then optimised by a simple reduction in flow rate; this may also require some slight changes to the mobile phase or temperature in order to maintain fast separation methods.


An example of the improvement in speed, cost and sensitivity gained by using short columns with narrow internal diameter are


injection volume was maintained at 2µl for each column. The lower flow rates also permit direct connection to the MS without flow splitting.


Conclusions Screening protocols provide a speedy answer to the question – which column, which mobile phase – in preparation for optimisation studies. The generic screens shown offer fast screening to achieve this. The effect on the screening protocol of injecting a composite set of chiral molecules using LC-MS may be minimal, affecting only sensitivity such that batch screening shows excellent potential for speeding up the column selection process further, without the need for use of parallel HPLC systems and producing methods that are immediately useful for MS detection. Further studies currently under way are hoped to lead to an MS protocol.


References 1.D.W.Armstrong, Y Tang, S Chen, Y Zhou, C Bagwell, J-R Chen; Anal Chem, 66 (1994) 1473-1484.


2.D S Bell, J E Claus, J Jones; Chirality 2009, Breckenridge, US , Poster P102 . Significant Improvements in ChiralMethod Development Using an LC-MS-Based Screening Approach, July 12 - 15, 2009.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60