Ultra-High Speed Analysis of USP Methods

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Kenichiro Tanaka, William Hedgepeth 　
Shimadzu Scientific Instruments, Inc., Columbia, MD
Ultra-high Speed Analysis of USP Methods
Conforming to New USP General Chapter 621 Allowed
Limits

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Introduction
The United States Pharmacopeia (USP) defines allowed adjustments of HPLC and GC
parameters in the general chapter 621 <chromatography>.
If the system suitability is met, method parameters can be changed within the allowed limits
without revalidation.
The general chapter 621 was revised in the first supplement to USP37-NF32 published on
February 1st
2014 and became official on August 1st
2014.
The feature of the new general chapter 621 is that a column packed with small particles can be
used if column length and particle ratio (L/dp) is kept constant between the designated and
modified column. This enables high speed analysis of USP methods more than ever.
Key parameters of allowed HPLC adjustments in the current and new general chapter 621 were
shown in Table 1 and 2, respectively.
The allowed adjustments for column length and particle size have been changed in the new
general chapter 621. This adjustment can be applied only for isocratic analysis.

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Introduction
Column length Can be adjusted by as much as ±70 %
Column inner diameter Can be adjusted if the linear velocity is kept constant
Particle size Can be reduced by as much as 50%, but cannot be increased
Flow rate Can be adjusted by ±50 %
Column length and
particle size
May be modified provided that the ratio of the column length (L) to
the particle size (dp) remains constant or into the range between
-25 % to +50 % of the prescribed L/dp ratio
Column inner diameter Can be adjusted if the linear velocity is kept constant
Flow rate Can be adjusted by ±50 %
Table 1: Allowed HPLC Adjustments in the Previous General Chapter
621 (official until July 31st
, 2014)
Table 2: Allowed HPLC Adjustments in the New General Chapter 621 (official
after August 1st
, 2014)

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Introduction
These changes are based on the theory that resolution can be kept constant as long
as L/dp is kept constant.
For example, if a method using a 150 mm L, 5 µm column (L/dp = 150,000 µm /5 µm
=30,000) is transferred to a method using a 50 mm L , 1.6 µm column (L/dp = 50,000
µm /1.6 µm = 31,250), similar resolution should be obtained.
Flow rate changes for both a change in column diameter and particle size can be
calculated by:
F2=F1 ×[(dc2
2
×dp1)/(dc1
2
×dp2)]
Where F1 and F2 are the flow rates for the original and modified conditions, respectively;
dc1 and dc2 are the respective column diameters; and dp1 and dp2 are the particle sizes.

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Materials and Methods
Reagents: Sulfacetamide, sulfanilamide, timolol maleate, glacial acetic acid, monobasic
sodium phosphate, and phosphoric acid were purchased from Sigma-Aldrich.
Water was made in house using a Millipore Milli-Q Advantage A10 Ultrapure Water
Purification System. Methanol and acetonitrile were purchased from Honeywell.
Standard solutions (Sulfacetamide analysis):
Mobile phase: Methanol, water, and glacial acetic acid (10:89:1)
Diluent: Methanol and 1% glacial acetic acid (10:90)
System suitability solution: 0.2 mg/mL of sulfacetamide and 0.05 mg/mL of sulfanilamide
in diluent. Sonicated for 5 min to dissolve
Standard solution: 0.2 µg/mL of sulfacetamide and 4 µg/mL of sulfanilamide in water
Reagents and Standards

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Standard solutions (Timolol maleate ophthalmic solution analysis):
pH 2.8 phosphate buffer: Dissolved 11.1 g of monobasic sodium phosphate in 1000 mL
of water, adjusted with phosphoric acid to a pH of 2.8, filtered and degassed.
Diluent: Prepared a mixture of acetonitrile and pH 2.8 phosphate buffer (2:1).
Mobile phase: Prepared a mixture of pH 2.8 phosphate buffer and methanol (65:35).
Standard preparation: Transferred 34 mg of timolol maleate to a 25 mL volumetric flask,
dissolved in and diluted with water to volume, and mixed. Transferred this stock
solution to a 50 mL volumetric flask, added 15 mL of Diluent, diluted with water to
volume, and mixed.
Reagents and Standards, Continued
Materials and Methods

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System
The standard solutions were injected to a Shimadzu Prominence system and
Nexera X2 system.
The Prominence system consisted of a LC-20AD pump, DGU-20A5R degassing
unit, SIL-20AC autosampler, CTO-20AC column oven, SPD-20AV UV-VIS
detector equipped with the conventional flowcell, and a CBM-20A system
controller.
The Nexera X2 system consisted of an LC-30AD pump, DGU-20A5R degassing
unit, SIL-30AC autosampler (loop injection mode with 20uL loop), CTO-30A
column oven, SPD-M30A photodiode array detector equipped with the standard
flow cell, and a CBM-20A system controller.

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Results - Adjustment of USP Method For
Speed
A USP method for sulfacetamide, which is a sulfonamide antibiotic, was transferred to a
UHPLC method.
The USP designates the use of a 4.6 × 150 mm, 5 µm, L1 (ODS) column and a 0.8 mL/min
flow rate for the organic impurities analysis of sulfacetamide. A 2.0 × 50 mm, 1.6 µm, L1
column can be used for adjustment of USP method for speed without method revalidation
because L/dp is kept constant.
The new flow rate after method transfer is calculated to 0.47 mL/min according to the
equation shown earlier. System suitability solution, standard solution, and sample solution
are designated to run in the monograph, but only the chromatogram of the system suitability
solution was shown in this poster.
The USP method and the ultra-high speed method were run on Prominence and Nexera X2
UHPLC system, respectively.
The analytical conditions are shown in Table 3, the analysis results are shown in Fig. 1, and
the system suitability results are shown in Table 4. As a result, analysis time and solvent
consumption were reduced to 1/10 and 1/15, respectively, with the system suitability
requirements met.

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Results
Parameter USP method Ultra-high speed method
System : Prominence Nexera X2
Column : Shim-pack VP-ODS Shim-pack XR-ODSIII
(150 mm L. x 4.6 mm I.D., 4.6 μm) (50 mm L. x 2.0 mm I.D., 1.6 μm)
Mobile Phase : See “Reagent and standards” Same as left
Flow Rate : 0.8 mL/min 0.47mL/min
Column Temperature : Ambient Same as left
Injection Volume : 10 µL 2 µL
Detection : UV 254 nm Same as left
Table 3: Analytical Conditions

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Results
Fig.1: Chromatogram of System Suitability Solution
(Upper: USP method on Prominence, Lower: Ultra-high speed method on Nexera X2)
0.00 0.25 0.50 0.75 min
0.0
0.5
1.0
1.5
2.0
2.5
mAU(x1,000)
0.0 2.5 5.0 7.5 min
0.00
0.25
0.50
0.75
1.00
mAU(x1,000)
■P eaks
1. Sulfanilamide,
2. Sulfacetamide
USP method (Prominence)
Ultra-high speed method (Nexera X2)
1
2
1
2

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Results
System suitability requirements
USP method
(Prominence)
Ultra-high speed
method
(Nexera X2)
USP resolution between sulfacetamide and
sulfanilamide
≥ 5.0 14.54 12.23
USP tailing factor for sulfacetamide ≤ 1.5 1.09 1.04
Relative standard deviation for sulfacetamide ≤ 2.0 %
Rt 0.015 % Rt 0.037 %
Area 0.067 % Area 0.103 %
Table 4: System suitability results

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Results
USP method on Nexera X2
A USP method for timolol maleate ophthalmic solution was run on both the Prominence and
Nexera X2 UHPLC system without changing the designated method parameters. The USP
designates the use of a 4.6 × 150 mm, 5 µm, L1 (ODS) column and a 1.2 mL/min flow rate
for the assay. The analytical conditions are shown in Table 5, the analysis results are
shown in Fig. 2, and the system suitability results are shown in Table 6. As a result, the
system suitability requirements were easily met on the UHPLC system, similar to a standard
HPLC system.
Parameter USP method on conventional HPLC USP method on UHPLC
System Prominence Nexera X2
Column Shim-pack VP-ODS Same as left
(150 mm L. x 4.6 mm I.D., 4.6 μm)
Mobile Phase See “Reagent and standards” Same as left
Flow Rate 1.2 mL/min Same as left
Column Temperature 40C° Same as left
Injection Volume 10 µL Same as left
Detection UV 295 nm Same as left
Table 5: Analytical Conditions

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Results
0.0 2.5 5.0 7.5 min
0.0
0.5
1.0
1.5
2.0
2.5
mAU(x100)
0.0 2.5 5.0 7.5 min
0.0
0.5
1.0
1.5
2.0
2.5
mAU(x100)
■P eak
1. Timolol
USP method on conventional HPLC
(Prominence)
USP method on UHPLC (Nexera X2)
1
1
Fig.2: Chromatogram of a Standard Preparation
(Upper: USP method on Prominence, Lower: USP method on Nexera X2)

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Results
System suitability requirements
USP method
(Prominence)
USP method
(Nexera X2)
USP tailing factor ≤ 2.0 1.12 1.11
USP column efficiency ≥ 3600 6354 6965
Relative standard deviation ≤ 2.0 %
Rt 0.027 % Rt 0.082 %
Area 0.034 % Area 0.062 %
Table 6: System suitability results

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Conclusions
A USP method was successfully transferred to an ultra-high speed method with the
system suitability requirements met.
As a result, analysis time and solvent consumption were reduced to 1/10 and 1/15,
respectively. Additionally, a USP method was run on the Nexera X2 UHPLC system
without changing method parameters.
The system suitability requirements were easily met on the UHPLC system, similar to
a standard HPLC system.
This result means that this system is also suitable for those who are running traditional
USP methods on a standard HPLC system and considering the adoption of a UHPLC
system for high speed analysis of USP methods in the future.

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References
1. USP General Chapter 621, USP 37-NF 32, First supplement
2. USP Monograph, Sulfacetamide, USP 37-NF 32, First supplement
3. USP Monograph, Timolol maleate ophthalmic solution, USP 37-NF 32, First
supplement

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