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LAAN-A-LC-E241

Application High Performance Liquid Chromatography

News Ultra-High Speed Analysis of USP Methods Conforming

to Permissible Limits in New USP General Chapter 621
No.L464

In the General Chapter <621> Chromatography of the This change is based on the theory that if L/dp is kept
U.S. Pharmacopeia (USP), the ranges within which constant, equivalent separation performance will be
changes to HPLC and GC parameters are permissible are maintained. For example, if a column of length 150 mm
indicated, and as long as the values are within that and particle size of 5 µm (L/dp = 150,000 µm / 5 µm =
range, and as long as the system suitability requirements 30,000) is changed to one of column length 50 mm and
are satisfied, the method can be changed without particle size 1.6 µm (L/dp = 31,250), L/dp can be
revalidation. An example of a faster method by which maintained at +4.2 %, thereby obtaining equivalent
these changes remained within the permissible range separation performance. In addition, the following formula
was previously introduced in Application News No. L448. can be used with respect to flowrate.
The General Chapter 621 was again revised, and the F2 = F1 × [(dc2 × dp ) / (dc1 × dp )]
2
2
revision has been in effect as of August 1, 2014. 1 2
Previously, a reduction of up to 50 % in the column F1 and F2 express the original USP monograph flowrate and
particle size was permitted, but according to the new the modified flowrate, respectively. Also, dc and dp express
revisions to the General Chapter 621 that has been in the column internal diameter and column packing particle
effect, the column particle size can be freely selected. size, respectively. This expression includes two principles.
Therefore, even though the current particle size of 5 µm The first is that the flowrate is adjusted in proportion to the
is specified, columns with particle size in the sub-2 µm column cross-sectional area, or in other words, the linear
region may be used, permitting even faster analyses. velocity is kept constant. The second is that because the
Here, we introduce an example of a faster analysis of a particle size and optimal flowrate are inversely proportional,
USP method in compliance with the new General the point is to change the flowrate in inverse proportion to
Chapter 621 using the Nexera X2 ultra high the particle size. For example, the USP monograph
performance liquid chromatograph and the Shim-pack stipulates that for a 4.6 × 150 mm, 5 µm column, a
XR-ODS Ⅲ high-speed analytical column. flowrate of 1.0 mL/min is specified, and if the column is
changed to a 2.0 × 50 mm, 1.6 µm column, a flowrate of
*Shimadzu Scientific Instruments, Inc., USA 0.59 mL/min is required. Further, if the particle size is
changed from ≧3 µm to ＜3 µm, it is permissible to increase
n Permissible Limits of Modification for HPLC Parameters the flowrate further as long as the column efficiency does
Tables 1 and 2 list the permissible limits of the HPLC not decrease more than 20 %. Conversely, if a ＜3 µm
column-related parameters according to the General particle size is changed to ≧3 µm, the flowrate may need to
Chapter 621 of the previous and new version of the be decreased so that the column efficiency does not
specifications, respectively. These changes apply to the decrease more than 20 %.
column length, particle size, and flowrate. It should be
noted that these changes apply only to isocratic n Speed Enhancement of a USP Method
analysis, and are not applicable to gradient analysis. Here, we introduce a faster method of the impurity analysis of
sulfacetamide as defined in the USP. Sulfacetamide is a type of
Table 1 Permissible Limits of HPLC Parameters in the Previous sulfonamide antibacterial drug. The column specified in the
General Chapter 621 (effective until July 31, 2014) USP for this analysis is the 4.6 × 150 mm, 5 µm, L1 (ODS),
using a flowrate of 0.8 mL/min. In this case, if the
Column diameter Can be adjusted if the linear velocity is kept constant 2.0 × 50 mm, 1.6 µm, L1, Shim-pack XR-ODS Ⅲ column is
Column length Can be adjusted by as much as ±70 % used, almost the same L/dp value is obtained. The flowrate can
Particle size Can be reduced by as much as 50 %, but cannot
be increased. be calculated as 0.47 mL/min from the formula just introduced.
Flowrate Can be adjusted based on column cross-sectional Although analysis of a system suitability test solution, a
area ratio. In addition, can be adjusted by ±50 %. standard solution, and the sample solution are all required in
an actual analysis, here we show the chromatograms obtained
Table 2 Permissible Limits of HPLC Parameters in the New from analysis of system suitability test solutions (0.2 mg/mL
General Chapter 621 (effective as of August 1, 2014) USP sulfacetamide reference standard, 0.05 mg/mL
Column diameter Can be adjusted if the linear velocity is kept constant sulfanilamide USP reference standard). For the HPLC system,
Column length and May be modified provided that the ratio of the the Prominence was used, and for the UHPLC system, the
particle size column length (L) to the particle size (dp) Nexera X2 was used. Table 3 shows the analytical conditions,
remains constant or within the range of -25 % Fig. 1 shows the chromatograms obtained, and Table 4 shows
to +50 % of the prescribed L/dp ratio.
Flowrate Can be adjusted using column cross-sectional the system suitability test results. Using the Nexera X2 system
area ratio and particle size inverse ratio.* and the Shim-pack XR-ODSⅢ column, in addition to meeting
When a change is made from ≧ 3 µm particle the system suitability requirements, we were able to shorten
size to ＜ 3 µm particle size, or from ＜ 3 µm
particle size to ≧3 µm particle size, the linear the analysis time significantly. In this investigation, not only was
velocity may be changed within a range in the analysis shortened to about 1/10 the time on a per-minute
which the column efficiency does not decrease
more than 20 %.* basis, solvent consumption was also reduced to about 1/15
In addition, it can be adjusted by ±50 %. that normally required.
* See text