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.

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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].

Table 1. USP column packaging L-groups, definitions, compliant separation columns from Metrohm, and the USP Monographs where these are applicable.

Packing	Description	Compliant Metrohm column	USP Monograph(s)
L46	Polystyrene/divinylbenzene substrate agglomerated with quaternary amine functionalized latex beads (diameter 9–11 µm).	Metrosep A Supp 1 - 250/4.6	Voriconazole, Sodium Fluoride and Acidulated Phosphate Topical Solution, Sodium Fluoride Oral Solution, <345> Assay for Citric Acid/Citrate & Phosphate
L76	Silica-based weak cation-exchange material, 5 µm in diameter. Substrate is surface polymerized polybutadiene-maleic acid to provide carboxylic acid functionalities. Capacity not less than 29 µEq/column.	Metrosep C 4 - 250/4.0 Metrosep C 6 - 150/4.0	Calcium Acetate Capsules, Potassium Citrate & Citric Acid Oral Solution, Potassium Bicarbonate Effervescent Tablets for Oral Solution, Potassium and Sodium Bicarbonates and Citric Acid Effervescent Tablets for Oral Solution, Potassium Bicarbonate and Potassium Chloride Effervescent Tablets for Oral Solution (Potassium)
L91	Strong anion-exchange resin consisting of monodisperse porous polystyrene/divinyl benzene beads (diameter 3–10 µm) coupled with quaternary amine.	Metrosep A Supp 10 - 250/4.0 Metrosep A Supp 16 - 100/4.0 Metrosep A Supp 17 - 150/4.0 Metrosep A Supp 19 - 150/4.0	Zinc Oxide, Zinc Oxide Neutral, Zinc Oxide Powder, Zinc Sulfate Ophthalmic Solution, <591> Zinc Determination, Sodium Fluoride, Sodium Fluoride Gel, Sodium Fluoride Oral Solution, Potassium Bicarbonate and Potassium Chloride Effervescent Tablets for Oral Solution, Sodium Fluoride Tablets, Stannous Fluoride, Sodium Monofluorophosphate, Stannous Fluoride Gel, Potassium Phosphates Compounded Injection, Sodium Chloride Tablets for Solution, Sodium Phosphates Compounded Injection
L97	Weak cation-exchange resin consisting of a highly cross-linked core of 5.5 µm porous particles having a pore size of 2000 Å and consisting of ethylvinylbenzene cross-linked with 55% divinylbenzene. Substrate is surface grafted with carboxylic acid functionalized groups. Capacity not less than 2400 µEq/column (4 mm x 25 cm).	Metrosep C Supp 2 - 250/4.0	Sodium Bicarbonate Compounded Injection, Sodium Phosphates Compounded Injection
L105	A strong anion-exchange resin consisting of highly cross-linked 9 µm supermacroporous (2000 Å) particles functionalized with very low cross-linked latex (0.2%) to provide alkyl quaternary ammonium ion sites.	Metrosep A Supp 4 - 250/4.0	Sodium Nitrite

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:

the user selects a column that belongs to the same L-group as specified for the fully validated method
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.

Figure 1. Illustrated schematic showing how equivalent separation columns are proposed, reviewed, approved, and accepted by the USP.

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).

Table 2. Requirements regarding column equivalency for Voriconazole Related Compound F as per USP.

Voriconazole Related Compound F as per USP - Summary Table
Parameters	USP Monograph	Metrohm
Eluent (Mobile phase)	Sodium hydroxide solution: 470 g/L of sodium hydroxide in water Mobile phase: methanol, water, and Sodium hydroxide solution (500:1500:0.175)	Same as USP Monograph
Chloride stock solution	86 μg/mL of sodium chloride in water	Same as USP Monograph
Standard stock solution	250 μg/mL of USP Voriconazole Related Compound F RS. Dissolve in 50% of the final volume with methanol and dilute with Mobile phase to volume.	Same as USP Monograph
Standard Solution	5.0 μg/mL of USP Voriconazole Related Compound F RS from the Standard stock solution in a mixture of methanol and Mobile phase (50:50)	Same as USP Monograph
System suitability solution A	5 μg/mL of USP Voriconazole Related Compound F RS from the Standard stock solution and 1.7 μg/mL of sodium chloride in a mixture of methanol and Mobile phase (50:50)	Same as USP Monograph
System suitability solution B	2.5 μg/mL of USP Voriconazole Related Compound F RS from the Standard solution in Mobile phase	Same as USP Monograph
Sample Solution	5 mg/mL of Voriconazole. Dissolve in 50% of the final volume with methanol and dilute with Mobile phase to volume.	Same as USP Monograph
Detection	Conductivity with suppression	Same as USP Monograph: Conductivity with suppression (MSM_ChS)
Column	4 mm × 5 cm guard column and 4 mm × 25 cm analytical column; both packing L46	Metrosep A Supp 1 Guard/4.6 (P/N: 6.1005.340) Metrosep A Supp 1 - 250/4.6 (P/N: 6.1005.300)
Flow Rate	1.0 mL/min	Same as USP Monograph
Injection Volume	20 µL	Same as USP Monograph
Run Time	not mentioned	60 minutes
Column Temperature	40 °C	Same as USP Monograph

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).

Table 3. Determination of the Metrohm IC system suitability as compared to the USP Monograph requirements for the analysis of Voriconazole Related Compound F (VRC-F).

System suitability (n=6)	USP Monograph	Metrohm
Tailing factor (System suitability solution B)	NMT 2.0 for VRC-F peak	1.219 (PASS)
Relative Standard Deviation (System suitability solution B)	NMT 10.0% for VRC-F ion	2.347% (PASS)
Resolution between VRC-F ion and Chloride ion (System suitability solution A)	NLT 3.5	11.41 (PASS)

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.

Table 4. USP acceptance criterion for the analysis of voriconazole with ion chromatography.

Voriconazole (Batch No: VZFP22009)	USP Monograph	Metrohm
Mean Sample results: Triplicate Sample preparations (Voriconazole Sample solution)	NMT 0.1%	0.04% (PASS)

Figure 2. Example chromatogram of a USP reference standard for Voriconazole Related Compound F determined with the Metrosep A Supp 1 column.

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.

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[16] Pfundstein, P.; Martin, C.; Schulz, W.; et al. IC–ICP-MS Analysis of Gadolinium-Based MRI Contrast Agents (TA-023); LCGC Asia Pacific, 2011.

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[18] U. S. Pharmacopeia/National Formulary. General Chapter, <621> Chromatography; USP-NF: Rockville, MD, USA, 2023. DOI:10.31003/USPNF_M99380_07_01

[19] U. S. Pharmacopeia/National Formulary. General Chapter, <1065> Ion Chromatography; USP-NF: Rockville, MD, USA, 2023. DOI:10.31003/USPNF_M897_01_01

[20] U. S. Pharmacopeia/National Formulary. General Chapter, <1225> Validation of Compendial Procedures; USP-NF: Rockville, MD, USA, 2023. DOI:10.31003/USPNF_M99945_04_01

[21] Schäfer, H.; Läubli, M. Monograph Ion Chromatography, 8.108.5077EN; Metrohm AG: Herisau, Switzerland, 2023.

[22] U. S. Pharmacopeia. Chromatographic Columns; USP: Rockville, MD, USA, 2023.

[23] U. S. Pharmacopeia/National Formulary. Reagents; USP-NF: Rockville, MD, USA, 2023.

[24] U. S. Pharmacopeia/National Formulary. <591> Zinc Determination. In General Chapter; USP/NF, Rockville, MD, USA; DOI:10.31003/USPNF_M99350_05_01

[25] ASHP. AHFS® Patient Medication Information™: Voriconazole, 2023.