Figure 7: Individual standard injections using step 6 with the Diamond Hydride column. Standard injections are (a) ascorbic
acid, (b) riboflavin, (c) pyridoxine, and (d) thiamine.
Selectivity issues such as this can often be solved with a simple change in the mobile-phase gradient program. It is best
to address inadequate or excessive retention and peak shape issues first before optimizing the gradient. A more gradual gradient
should provide additional selectivity between ascorbic acid and riboflavin, resulting in acceptable resolution between the
two peaks. Step 6 features the same mobile-phase solvents as step 5, but now uses a more gradual gradient slope. The data
for this method are shown in Figure 7. This method now addresses all the goals that were set for the analysis. The retention
range of the analytes is within a suitable range and the critical peak pair is baseline-resolved.
Figure 8: Retention times obtained for each of the four analytes with each of the six method steps (see Tables I and II for
method conditions).
To summarize the process of this method development strategy, Figure 8 shows a comparison of the retention times for each
analyte using the six method steps that were discussed. At each step, the data is more refined and more adequately approaches
the method goals:
Step 1: Low retention for most analytes. Different retention mode needed.
Step 2: No retention for one analyte. Suggested use of a different mobile-phase additive.
Step 3: Excessive retention for one analyte with a broad peak shape. Suggested a combination of the two additives would allow
for a total separation.
Step 4: Good retention for three of four analytes. Lower retention than expected, suggested insufficient buffering capacity/ionic
strength.
Step 5: All analytes retained well. Co-elution of two analytes.
Step 6: All analytes well retained and well resolved.
Figure 9: LC–MS extracted ion chromatograms (EICs) from a mixture of the four test solutes using step 6 method conditions
with the Diamond Hydride column. EICs are (a) ascorbic acid (177.0394 m/z [M + H]+), (b) riboflavin (377.1456 m/z [M + H]+),
(c) pyridoxine (170.0812 m/z [M + H]+), and (d) thiamine (265 m/z [M – 2Cl – H]+).
After the final method had been devised, transfer of the HPLC–UV-based methodology to LC–MS was investigated. A mixture of
the four test solutes was prepared and injected using step 6 method conditions with an LC–MS system and the low carbon-bonded
silica hydride column. Figure 9 shows the extracted ion chromatograms (EICs) obtained from the injection. Three of the ions
are present as [M + H]+ , but thiamine is observed as [M – 2Cl – H]+ (9). This data demonstrates how the method that was developed with HPLC–UV can be successfully transferred to an LC–MS system.
Because HILIC methods are sometimes reported to be prone to a lack of precision or robustness, it was important to study whether
the ANP method that was developed shares similar disadvantages. For example, ANP theory predicts that retention time precision
should be excellent because a large and variable water layer is not involved in the retention mechanism. In addition, robustness
studies can provide insight into which variables affect the chromatography most significantly.
Table III: Intra-* and interday† intermediate precision of analyte retention times (%RSD) using step 6 method conditions
To study the method's intermediate precision, intra- and interday repeatability studies were conducted. Injections of the
mixture were made five times a day over the course of three different days. Percent relative standard deviation (%RSD) of
analyte retention times for both intra- and interday repeatability are shown in Table III. The low %RSD values obtained in
each case illustrate how ANP retention is capable of producing reliable and consistent results.
Figure 10: Differences in retention times ΔtR obtained from original step 6 method parameter values and the low (-) and high
(+) adjusted values given in Table IV: (a) ascorbic acid, (b) riboflavin, (c) pyridoxine, and (d) thiamine.
To investigate the robustness of the method, slight but deliberate changes were made to several parameters of step 6 method
conditions. The investigated parameters are given in Table IV. Using the altered methods, the retention times of each analyte
were then compared to the original method values obtained on the same day. The results of the robustness study are shown in
Figure 10. The relative significance of each method parameter confirms what was discovered during method development. Ascorbic
acid and thiamine show the most deviation of the four analytes because of pH changes in Figure 10, as was observed in data
from steps 2 and 3. The pH gradient explains the higher deviation for the hold time %B and flow rate as well. Also, the directions
of the trends are consistent with what was observed in method development. For example, ascorbic acid and thiamine retention
increased with increasing mobile-phase pH.
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