製薬業界におけるイオンクロマトグラフィの応用

イオンクロマトグラフィ（IC）は、イオン性および極性のある分析物を分離・定量するための分析技術です。ICは水質や化学分析など、さまざまな用途で広く受け入れられ、製薬業界や規制機関での利用が拡大しています。近年では医薬品の分析にも適用されるようになりました。医薬品およびバイオ医薬品製品の標準化を推進するアメリカ薬局方-国家医薬品集（USP-NF、アメリカ薬局方（USP））は、近代化の取り組みを通じてICの可能性を高め、一般章、分析法、及び不純物試験において適切かつ有効な技術として位置づけました。

このコラムでは、イオンクロマトグラフィの原理を簡単に紹介した後、USP一般章<1065>「イオンクロマトグラフィ」を基に、製薬分析におけるICの応用と利点について紹介しています。

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• イオンクロマトグラフィの原理
  
• W製薬業界におけるイオンクロマトグラフィの重要性
• USP <1065> に基づくICの検出技術と応用

イオンクロマトグラフィの原理

イオンクロマトグラフィ（IC）は、高性能液体クロマトグラフィ（HPLC）の一種で、イオン性および極性のある分析物を同時に分離・定量するために使用されます。分離機構として、主にイオン交換が用いられます【1–3】（アニメーション1）。

イオン交換分離カラムと導電率検出器の組み合わせが、ほとんどのICアプリケーションで使用されていますが、UV/VIS検出器、アンペロメトリック検出器、または質量分析計との連携など、他の検出技術を使用することも一般的です【3,4】。液体クロマトグラフの主な構成要素は図1に示されています。これには、移動相（溶離液）の貯蔵を備えた高圧ポンプ、インジェクター（サンプル導入）、分離カラム（分析カラム）、および検出システム（誘導化、データ収集、データ処理を含む）が含まれます。

メトロームでのイオンクロマトグラフィの開発や、その多彩な検出技術については、以下の関連資料で詳しく紹介しています。

Blog post: History of Metrohm IC – Part 1

Monograph: Ion Chromatography

Monograph: Practical Ion Chromatography – An Introduction

製薬業界におけるイオンクロマトグラフィ（IC）の重要性

HPLC（高性能液体クロマトグラフィ）が製薬業界における液体クロマトグラフィの主流である一方、イオンクロマトグラフィ（IC）も広く受け入れられるようになりました。ICが他の液体クロマトグラフィや、滴定などの従来の手法に対して製薬業界で持つ利点は、以下のようにまとめられます【5,6】：

• サンプル中の複数成分を一度に測定
• イオン性および極性分析物専用の分析手法
• 非金属の流路
• 溶離液は塩類、弱酸、弱塩基に基づく
• サプレッション技術（アニメーション2）：低ノイズのベースラインを実現し分析感度を向上
• 柔軟な装置構成： 導電率、UV/VIS、アンペロメトリック検出、または質量分析計との連携など、多様な検出器に対応
• 高度な自動化による効率の最大化と所有コストの低減（例：インラインサンプル調製オプションや単一標準物質による校正）

IC as a validated approach for USP-NF standards

As one of the driving bodies for the standardization of pharmaceutical methods, the USP-NF ensures the quality, safety, and efficacy of pharmaceuticals within the manufacturing process and their use [6,7]. The history of USP-NF standards is quite long—over 200 years [6]. However, new quality criteria and technologies supported a global modernization initiative of the FDA (U.S. Food and Drug Administration) and USP-NF in 2010.

IC, as a methodological approach for analysis of trace impurities, excipients, active pharmaceutical ingredients (APIs), metabolite and degradation products, and ionic components in pharmaceutical solutions and body fluids [5,8–15], became a validated approach in numerous USP chapters, assays, impurity and identification tests. Here, IC partially replaced traditional methods (e.g., titration) or was included in new standards focusing on using state-of-the-industry methods.

Fundamentals of chromatographic techniques are given in the USP General Chapters <621> and <1065> [16,17]. These General Chapters describe details about the methodology and the overall setup, as well as key parameters and requirements for the accuracy and reliability of chromatographic methods in pharmaceutical analysis. 

USP General Chapters <621> and <1065>: Core principles and fundamentals for chromatography

USP General Chapter <621> gives an overview of several chromatographic techniques along with definitions of the apparatus and procedure (e.g., mobile phase, column, types of elution, method procedure), common parameters, and requirements for system suitability testing (e.g., system repeatability, system sensitivity, and peak performance) [16]. Chromatographic column classification is also explained with a list of packings (L-numbers), phases (G), and supports (S), functioning as references for chromatographers [18].
 

L-numbers for ion chromatographic USP approaches and insights about the column equivalency procedure are explained in the article below.

Blog post: Applying USP validated methods for separation column equivalency

On the other hand, USP General Chapter <1065> provides a comprehensive overview of IC [16,17] and its importance as an acknowledged test procedure for identifying and quantifying specific analytes (assays and impurities). Ion chromatography is a valid characterization procedure in many circumstances and aligns with all aspects of pharmaceutical production [8,17]. Its application field is broad, including quality control of raw materials, drug substances, or formulated products, as well as for assessing process waters used for manufacturing, culture media, cleaning solutions, or wastewater streams [17]. USP <1065> comprises basic knowledge of this technique: what ion chromatography is, instrumentation details (Figure 1), common detection mechanisms, procedures, and important notes for appropriate method development.

More extensive explanations of criteria and requirements for method validation are linked to General Chapter <1225> Validation of Compendial Procedures. This includes system suitability testing to ensure that the IC system functions properly before analysis by evaluating parameters including resolution, retention time, and peak shape, as well as requirements for validation with key parameters like linearity, accuracy, precision, specificity, limit of detection (LOD), and limit of quantitation (LOQ) [19]. In general, when developing an IC method, it is highly important to select appropriate mobile and stationary phases (Figure 1) to ensure efficient separation and appropriate selectivity and sensitivity (requirements as per USP <1225>) [17,19]. Mobile phases typically consist of diluted acids, bases or salts dissolved in high-purity water, while stationary phases can be either silica-based or polymer-based materials.

Due to their importance, columns and appropriate mobile phases are embedded in the monographs and General Chapters of USP. However, column equivalency gives users some freedom for implementing the method in the analytical workflow. 

Learn more about IC columns in our blog post series, starting here.

Blog post: Best practice for separation columns in ion chromatography (IC) – Part 1

IC detection techniques and their applications according to USP <1065>

Four commonly used IC detection methods are discussed in this section (click below to jump to a topic):

• conductivity detection
• UV/VIS detection
• amperometric detection
• IC hyphenated to mass spectrometry

Conductivity detection

The most common IC detection method is conductivity [17]. The principle – based on conductometry – is shown in Animation 3 [2]. Conductivity detection measures how well the ions in a solution conduct electrical current between two electrodes, providing information about the concentration of ionic species present. When combined with a suppressor, the background signal is reduced, and the sensitivity and signal-to-noise ratio are significantly enhanced (Animation 2) [2,17].

IC with conductivity detection has a wide application range for pharmaceutical analysis, including:

The particularity of IC lays in the determination of multiple analytes in one run as shown, e.g., for multiple anionic analytes (acetate, chloride, citrate, and sulfate in an infusion solution; AN-N-051) or for cationic components (sodium, potassium, calcium, and magnesium in drip feeding formula; AN-C-022). Thanks to high-capacity IC columns, separation of analytes is possible even in the strongest of matrices [7].

UV/VIS detection

UV/VIS – an optical detection technique which can be used directly, indirectly, or with post-column derivatization – is useful for detecting ions that absorb UV/VIS light [2,17].

Direct UV-VIS detection is used alongside conductivity detection, e.g., for the determination of ions which strongly absorb in the UV range (nitrite, nitrate, organic anions) to improve their detectability in the presence of high concentrations of inorganic ions (chloride, phosphate, sulfate) which either have no or only little UV absorption capability [2]. The principle of this detection method is shown in Animation 4.

For indirect UV detection, eluents with high absorbance in the visible or ultraviolet spectral region are used (e.g., phthalate buffers) [17]. The detection wavelength is selected such that the eluent absorbs, but the sample ions do not. This choice results in negative peaks which are proportional to the analyte concentration.

Amperometric detection

Amperometric detection is an electrochemical analytical method where the current generated by the oxidation or reduction of analytes at a working electrode is measured as a function of time [20] (Animation 5). 

A prerequisite is that the target ions must be able to either be reduced or oxidized, i.e., electroactive substances [2,17]. Example analytes are organic compounds, transition metals, anions (e.g., nitrite, nitrate, sulfide, sulfite), or carbohydrates [2]. Some examples of using IC with amperometric detection for the pharma industry include lactose (in dairy products and supplements, AN-P-089), catecholamines (in a pharmaceutical injection solution, AN-P-053), gentamicin (in gentamicin solution, AN-P-080), and propylene glycol (in diclofenac topical solution, AN-P-076).

Our White Paper describes the determination of polyribosylribitol phosphate (PRP) in a Haemophilus influenzae vaccine using IC with pulsed amperometric detection (Figure 4). 

The black line in the chromatogram shows a standard with a concentration of 3 µg/mL PRP. The blue-green line indicates the total PRP content (final concentration 19.42 mg/L), within 80–120% of the claimed label (16–24 mg/L). According to the quality criteria, free PRP is required to be less than 20% of the total PRP content. The sample (lime green line in the chromatogram) shows the determined free PRP content (2.03 mg/L final concentration) and fits within the requirements.

IC hyphenated to mass spectrometry

Ion chromatography can be hyphenated with mass spectrometers to enhance the detection sensitivity for organic acids, carbohydrates, or trace elements. The inert instrumentation ensures flexibility regarding the used mobile phase and protects against contamination, which is especially important for trace elemental speciation. The IC suppression technique ensures that the liquid which enters these highly sensitive mass spectrometers only contains analytes and water (and maybe some organic modifiers). This is beneficial for stable running conditions, analysis sensitivity, and extending the instrument’s lifetime.
 

Learn more about hyphenation of IC with MS with our resources here.

Hyphenation of ion chromatography and mass spectrometry

White Paper: An introduction to ion chromatography mass spectrometry (IC-MS)

Conclusion

Ion chromatography is a powerful, versatile analytical tool for pharmaceutical applications. It is capable of handling a wide variety of sample types and provides high sensitivity when detecting ionic and ionizable substances. IC’s ability to couple different detection strategies makes it invaluable in pharmaceutical manufacturing and for quality control.

Your knowledge take-aways

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

Flyer: Bring your USP methods up to date!

Blog post: Applying USP validated methods for separation column equivalency

White Paper: When HPLC fails: IC in food, water, and pharmaceutical analysis

On-demand webinar: From APIs to impurities and excipients – Ion chromatography for pharmaceutical analysis

On-demand webinar: Ion chromatography for pharmaceutical applications

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