200 / 658
6
The Utilization of Novel Platform in a LC-MS/MS Work ow for the Analysis of Vitamin D, Testosterone, Immunosuppressants, Chemotherapeutics and Cortisol
Conclusion
A large number of compounds, with logP values ranging from -1 to 5, have been validated on
a new LC/MS/MS platform demonstrating the viability of the Prelude SPLC System for
compounds of interest to clinical researchers..
The Prelude SPLC Systemʼs lower void volume results in sample run times that are 20-30%
shorter. The reduced run time results in reduced cost due to lower consumption of mobile
phases and less waste disposal.
The Prelude SPLC uses a single syringe fill per sample, which removes the need for pulse
dampeners, reduces the mechanical wear and tear on pump parts such as pump seal and
active check valves, and does not need proportioning valves. The result is far less required
maintenance, reducing operating cost and down time.
OQ for Each Compound
1.0
1.5
2.0
2.5
3.0
Cortisol
Time (min)
1.0
1.5
2.0
2.5
3.0
Methotrexate
Time (min)
1.0
1.5
2.0
2.5
3.0
25-Hydroxy Vitamin D3
Time (min)
1.0
1.5
2.0
2.5
3.0
Sirolimus
Time (min)
1.0
1.5
2.0
2.5
3.0
Everolimus
Time (min)
specificity criterion. Recoveries, including matrix effects, were all around 90% or higher. All the
data is summarized in Tables 1 to 3. Figure 1 depicts representative standard curves for each
compound tested. Representative chromatograms at the lower limit of quantitation (LLOQ) for
each compound are shown in Figure 2.
The improvement in run times resulting from the lower void volumes of the Prelude SPLC
System verses a conventional HPLC is illustrated in Figure 3 for vitamin D. The same mobile
phases and columns were used for the comparison. When using on-line clean-up the duration
of certain steps cannot be changed because they are dependent on the chromatographic
separation needed. The duration of others steps in the process are related to how long it takes
for solvent changes to reach the column. The sample clean-up and sample elution steps are
dependent on the chromatography and; therefore, the time for those steps remain the same.
However, the transfer, column cleaning and re-equilibration steps can be reduced. On a
conventional HPLC the transfer step was 75 sec vs. 60 seconds on the Prelude SPLC. The
column clean-up and equilibration steps were reduced from 150 to 60 seconds. The result is a
reduction in run time of 29% (5:15 minutes to 3:45 minutes). A shorter run time also reduced
solvent consumption by 33%.
FIGURE 3. Comparison of the Method Run Time for Vitamin D on a Prelude
SLPC LC/MS/MS System to that of a Conventional HPLC System
0
50
100
150
200
250
300
350
Prelude SPLC
Conventional HPLC
Sample
Clean-up
Sample
Clean-up
Sample
Transfer
Sample
Transfer
Sample Elution
Sample Elution
Column
Clean-up
Column
Clean-up
Equilbration
Equilbration
Time (sec)
1.0
1.5
2.0
2.5
3.0
Docetaxel
Time (min)