Applying USP validated methods for separation column equivalency

The United States Pharmacopeia (USP) started updating around 4300 Monographs and 220 General Chapters with modern technology in 2010 [1]. During this time, ion chromatography (IC) also became a more popular analytical technique as it matured. Regulatory agencies now consider IC as an acceptable technology to analyze pharmaceuticals and biopharmaceuticals. 

The scope of IC analyses in the pharma industry is quite broad. IC can assess trace impurities, excipients, active pharmaceutical ingredients (APIs), and metabolites, as well as determine ionic components in pharmaceutical solutions and bodily fluids [2–10]. These quality control parameters can be measured in raw materials, during the production process, and in final products. IC is also suitable for quality control of process water and wastewater – safeguarding production and ensuring that discharged wastewater meets regulations [2,11–17]. 

This blog article focuses on the incorporation of IC during the modernization of USP methods. Separation column equivalency is discussed, and how it makes labs more flexible by implementing alternative columns for analytical methods in accordance with USP requirements.

Click to go directly to a topic:

• USP General Chapters and Monographs
• USP guide to chromatographic validation, qualification, and system suitability
• Specifications and requirements for high-quality pharmaceutical analysis 
• The analytical column: an essential part of the methodology
• USP method acceptance criteria for columns
• Successful column equivalency studies by Metrohm
• The importance of column equivalency for pharmaceutical analysis

USP General Chapters and Monographs

The USP provides quality standards for safe drugs, pharmaceutical substances, dietary supplements, and food products. Basic assumptions and definitions are summarized in «General Notices and Requirements» chapters. «Monographs» and «General Chapters» describe test procedures and acceptance criteria or general guidance to ensure the identity, quality, and purity of products. 

USP guide to chromatographic validation, qualification, and system suitability

General Chapter <621> [18] (as well as <1065> [19] and <1225> [20]) focuses on validation and qualification of chromatographic methods, including IC. It also sets the requirements and framework for system suitability testing.

System suitability tests are an integral part of the analytical procedure. These tests ensure adequate performance of the chromatographic system. Compliance with the system suitability criteria is mandatory throughout a chromatographic procedure. A sample analysis is only acceptable if the suitability of the system has been demonstrated. 

Specifications and requirements for high-quality pharmaceutical analysis 

The overall validation and qualification process for a USP method to determine the analytes described in the Monographs and General Chapters is very complex and underlies strict guidelines as already mentioned. 

The general chromatographic procedures specified in these USP methods involve numerous details about the chromatographic system and conditions. For these conditions, the system suitability tests show that the chromatographic method is robust and valid to quantify the analytes of interest. Such tests include repeated sample and standard measurements which must fit the acceptance criteria as well as fulfill method performance criteria [18,21] as explained below: 

• number of theoretical plates (N, a measure for column efficiency)
• tailing factor (also called symmetry factor A_S, a measure for peak asymmetry) 
• resolution (R_S, describes the separation quality) 
• relative standard deviation – a measure of the system repeatability calculated from consecutive series of measurements for not fewer than three, but up to six injections or applications of a reference solution

Modern chromatographic software programs (e.g., MagIC Net) facilitate the overall calculation procedure of these parameters. However, the complete validation procedure is strict, laborious, and very complex.

The analytical column: an essential part of the methodology

Considering the methodological parameters, it is evident that the column is a key part of the method. This is specified by the dimensions and the column packing (or L-group classification as per the USP database). 

Table 1 lists some L-groups relevant to IC analysis along with their description and equivalent Metrohm column examples. These are found in the USP column database [22] and are also specified in the USP documents chapter «Reagents» [23].

USP method acceptance criteria for columns

First, the column must be assigned to a specific L-category. The materials for each L-group or category have certain properties and column physiochemistry (see Table 1 for examples). These need to match the properties and column chemistry of the separation column in question. L-group categories may include different columns from a single provider or from different manufacturers. 

If the USP assigns an individual column to an L-group, then the conditions described in a Monograph can be qualified (e.g., zinc oxide determination according to USP <591> [24]; AN-U-076) as described earlier. The L-group is specified for each IC method—not a particular column. This allows some user flexibility depending on their preference when selecting a column of the same L-group for method qualification (Table 1). 

Column equivalency

Column equivalency now allows laboratories and manufacturers to qualify columns for validated chromatographic methods. Figure 1 shows the general procedure. 

There are two options to perform column equivalency testing without the need for a full validation:

1. the user selects a column that belongs to the same L-group as specified for the fully validated method
2. the user selects a column that belongs to an L-group similar in its physicochemical characteristics of the stationary phase along with no change of the identity of the substituent

USP General Chapter <621> [18] specifies the requirements for this procedure along with the suitability and acceptance criteria of the validated method for which the column needs to be qualified. 

Successful column equivalency studies by Metrohm

Metrohm is involved with performing full USP method validations. One example is described in the Application Note AN-S-398: «Phosphate in sodium and potassium phosphates compounded injections». Here, the Metrosep A Supp 17 - 150/4.0 column is used for the determination of phosphate. 

Several Metrohm columns (Table 1) were qualified for USP methods within the column equivalency process. Other examples include the validation of the Metrosep A Supp 4 - 250/4.0 column for the assay of nitrite in sodium nitrite (AN-S-400) or the Metrosep A Supp 1 column for quantification of voriconazole as shown in more detail below.

Application example: analysis of voriconazole

The Metrosep A Supp 1 column has been qualified by the USP for analysis of Voriconazole Related Compound F. Voriconazole is a medication used to treat serious fungal infections [25]. Table 2 lists all the USP requirements and method parameters as well as the achieved performance characteristics for the column equivalency study. 

The study was performed with a 940 Professional IC Vario system using Voriconazole Related Compound F RS solutions. Different concentrations (2.5–5 µg/mL) and compositions were used for system suitability testing (Table 3). Additionally, a sample voriconazole solution was tested (Batch No: VZFP22009, Table 4). 

The Metrohm IC system using the Metrosep A Supp 1 - 250/4.6 column met the USP suitability criteria for the analysis of Voriconazole Related Compound F RS (Table 3).

This study also met the acceptance criterion for the voriconazole sample analyses (Table 4) and was accepted by USP. An example chromatogram of Voriconazole Related Compound F is shown in Figure 2.

The importance of column equivalency for pharmaceutical analysis

Dedicated laboratory ion chromatography systems may differ from USP-validated setups. This is mostly due to the chromatographic columns in use. The USP Monograph modernization initiative has broadened the column selection to researchers. This is highly beneficial for the pharmaceutical industry.

Column equivalency studies allow alternative analytical columns to be used for high-quality pharmaceutical analysis. This saves a significant amount of time and effort by avoiding a full method validation procedure of several weeks. Ultimately, the accuracy and reliability of the analytical results are still achieved as per the validated method. 

Metrohm offers several USP-validated applications as well as alternative columns for validated USP methods approved by successful column equivalency studies.

Your knowledge take-aways

Drug monographs, assays, and impurity evaluations – Bring your USP methods up to date with Metrohm Ion Chromatography

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On-demand webinar: Analysis of impurities in pharmaceuticals using ion chromatography