Analysis of Small Organic Acids by Capillary Electrophoresis

Small organic acids such as acetic, citric and lactic are ideal candidates for analysis by capillary electrophoresis (CE) because they are small and highly charged. Methods have been developed and validated for a range of applications and are in routine use in a number of industries. This article covers the applications, separation conditions and reasons for using CE.

What Are These Small Organic Acids?

They are simple low-molecular-weight non-aromatic carboxylic acids. Examples include citric, maleic, succinic, lactic, acetic and tartaric acid. These species are ideal candidates for analysis by capillary electrophoresis (CE) because they are small and highly charged and a number of applications have been developed which are in routine use in many industries. A comprehensive review of the subject area is available in reference (1).

Why Use CE?

The methods are relatively fast with typical analysis times of 5 min. Kits are available from a number of suppliers and contain pre-prepared reagents and standards which provide reproducible separations. The separations are performed on standard CE equipment and capillaries which may prevent the need to purchase specific equipment and consumables such as ion-exchange chromatography systems and columns. The capillaries can be rinsed between samples which allows direct injection of liquid samples and can reduce the need for sample clean-up prior to analysis.

How Are They Detected?

There are a number of detection approaches used (1).

Indirect UV Detection: This is the most frequently used detection mode. An additive is included in the electrolyte which migrates at the same speed as the acids and provides the background signal for indirect detection. It is important that the additive moves at a similar rate to obtain good peak symmetry and improved detection limits. Examples that are frequently used are phthalate (2) and PDC (2,6-pyridinedicarboxylic) (3). Additive concentrations are typically in the order of 5–10 mM to give optimum sensitivity. Zwitterionic buffers such as MES (morpholinoethanesulphonic acid) are used (2) in indirect ultraviolet (UV) detection methods because they provide good buffering capacity with low operating currents. High currents lead to high temperatures within the capillary which generates refractive index changes and poor baseline noise.

Direct UV Detection: The organic acids have a limited amount of conjugation which allows them to be directly detected at low wavelengths. This is achieved using inorganic buffers such as phosphate or borate that have no residual UV absorbance. Enhanced detection is possible (4) using wavelengths as low as 185 nm. In some cases, diode array detection (DAD) is used (5) to enhance sensitivity and eliminate interference from co-migrating species, for example 200 nm is used as the primary wavelength with 260 nm as the reference wavelength.

Pre-separation derivatization of the organic acids is also possible to enhance sensitivity. For example, acids were derivatized with 2-nitrophenylhydrazine and determined with UV detection at 230 nm (6).

Mass-Spectrometry: Capillary electrophoresis-mass spectrometry (CE–MS) has been a routine combination because robust and reliable interfaces are available. Negative ion mode detection was used to determine a range of organic acids and amino acids in metabolic studies (7). This was applied specifically to pineapple leaves to study acid metabolism. CE–MS has also been used for analysis of organic acids in biological samples (8).

Conductivity Detection: Contactless conductivity detectors are commercially available and these have been applied to the analysis of organic acids in biological samples (9). This is more of a niche detection mode.

What Separation Conditions Are Used?

Often cationic surfactants are added to the electrolyte to reverse the electroendosmotic flow (EOF) by forming a double layer and a positive charge on the capillary surface. The EOF moves in the same direction as the negatively charged acids and gives good peak shape. Typically, the cationic surfactant used (2) is tetradecyltrimethylammonium bromide (TTAB) with concentrations in the region of 0.5 mM.

Selectivity can be optimized through pH adjustment of the electrolyte because 0the acids have multiple pKa values. For example, an optimal separation of tartaric, citric, succinic and acetic acids was obtained using a pH of 5.2 (2).

Selectivity additives can also be used. For example, 0.24 mM CaCl₂ was used to optimise selectivity (10). The organic acids selectively chelate/interact with the organic acids which alters the separation and can be used to enhance resolution.

What Are The Applications?

Clinical Analysis: This is a popular application as CE analysis of organic acids can be used to study and/or monitor different diseases such as diabetes, diseases in newborns or metabolism disorders. A recent review (8) summarizes the methodologies and applications.

A specific method (9) allowed separation of 29 organic acids occurring in urine. The optimized analytical system used a background electrolyte of 20 mM 2-morpholinoethanesulphonic acid (MES)/NaOH+10% (v/v) methanol, pH 6.0 (pH is related to the 20 mM MES/NaOH buffer in water). The repeatability of the procedure developed is characterized by the coefficients of variation, which vary between 0.3% (tartaric acid) and 0.6% (5-hydroxy-3-indoleacetic acid) for the migration time and between 1.3% (tartaric acid) and 3.5% (lactic acid) for the peak area. The procedure permitted quantitation of 20 organic acids in a real urine sample and was applied to monitoring of the occurrence of the inborn metabolic fault of methylmalonic aciduria.

Food Analysis: A rapid CE method with direct UV detection has been used (10) to determine the most important non-aromatic organic acids in honey with a really simple treatment of the sample. Oxalic, formic, malic, succinic, pyruvic, acetic, lactic, citric and gluconic acids were determined with an analysis time of 4 min. The electrolyte composition was phosphate as the carrier buffer (7.5 mM NaH₂PO₄ and 2.5 mM Na₂HPO₄), 2.5 mM tetradecyltrimethylammonium hydroxide (TTAOH) as electroosmotic flow modifier, and 0.24 mM CaCl₂ as selectivity modifier, with the pH adjusted at 6.40 constant value. The method was fully validated and applied to real samples. The advantages of the method were described as sample preparation simplicity, short analysis times and low consumption of chemicals. The method is in routine use.

Silage is grass stored for animal feed. A process of fermentation occurs during storage. Organic acids and some inorganic anions may have an affect on the nutritive quality of silage. Nine organic acids (formate, acetate, propionate, butyrate, n-valerate, iso-valerate, n-hexanate, citrate and lactate) and three inorganic anions (nitrate, nitrite and chloride) were determined by CE (3). Samples were extracted with water, purified through a cartridge column to remove interferences and diluted with water. A 30 mM 2,6-pyridinedicarboxylic acid (PDC) solution was used as a background electrolyte (BGE) at pH 12 with 0.5 mM of cetyltrimethylammonium hydroxide (CTAH). The method was fully validated by a collaborative study involving five laboratories. Various samples were analysed and it was determined that nitrate and citrate levels decreased over a long-term storage period of nine months. The acetate and lactate contents were higher in the high-moisture silage than the low-moisture silage.

Malic, citric, succinic, pyruvic, acetic and lactic acids effect were determined in beer samples (5). A pH 7 sodium hydrogen phosphate buffer was used with direct UV detection. An analysis time of 4 min allowed rapid determination of these acids. Analysis of beer samples gave good agreement with reference methods.

Eleven organic acids in a range of beverages (wine, beer and fruit and vegetable juices) have been determined by a CE method (6). The analysis involved a pre-separation derivatization with 2-nitrophenylhydrazine and detection at 230 nm. A pH 10 borate buffer containing 10% acetonitrile was found to give the optimum separation within 12 min. The method was fully validated with recovery data of 97%. Data from the CE method compared well with reference methods and literature values.

Wine Analysis: Organic acid content in wines is an important application because the content greatly affects the taste. This is a routine application for CE and a number of papers have been published showing good data. For example, a number of Brazilian wines have been profiled using a CE method with indirect UV detection (11). Sample preparation involves a simple 1:5 dilution with water and direct injection. Indirect UV detection was used and the method was fully validated with recoveries of 96–102% Tartaric, malic, lactic, succinic, acetic and citric acid content were determined with a run time of 5 min.

A CE method (OIV-MA-AS313-19: Organic acids and sulphates by capillary electrophoresis) is included (12) in the compendium of international methods for wine analysis: http://www.oiv.int/oiv/info/enmethodesinternationalesvin. The method allows quantitation of a range of organic acids and sulphate with chlorate used as an internal standard. The method was validated including a inter-laboratory repeatability exercise across five laboratories. Examples of the separations can be accessed from the official website.

Pharmaceutical Analysis: Basic drugs are usually manufactured as salts. The choice of counter-ion greatly affects the pharmaceutical performance of the compound because it influences properties such as solubility, crystallinity and stability. Typical inorganic counter-ions include chloride and phosphate. Organic acid counter-ions include maleate, succinate and citrate. There is a requirement that the counter-ion content is measured in each drug batch. CE is widely used for this measurement.

An example of this is the measurement of succinate content in sumatriptan succinate (a migraine treatment) (2). Separation conditions were a 27 cm × 75 μm capillary (Composite metal services, Harrow, UK), 3.0 s injection, 0.5 mM TTAB/5.0 mM phthalate/50 mM MES pH 5.2, 30 C, -3 kV applied voltage with indirect UV detection at 254 nm. Citrate was used as an internal standard and the method fully validated. The method gave good precision; for example, 10 replicate calibration preparations gave a relative standard deviation (RSD) 0.18% for response factors. Robustness of the method was successfully assessed using experimental design. The factors studies were pH, [MES], injection time, [TTAB], [phthalate], voltage and temperature. Ten replicate samples gave results for succinate content (%w/w) of 28.589, 28.624, 28.808, 28.408, 28.396, 28.919, 28.664, 28.682 28.673 and 28.712% w/w with an RSD of 0.42% and a mean of 28.642% The theoretical succinate content is 28.573%

Sodium caprylate and similar compounds such as acetyltrytophan are used as stabilizers/preservatives in human albumin solutions to protect the albumin from aggregation or heat induced denaturation. Citrates may be present in these solutions. CE methods have been developed and validated (13) for these analyses using indirect UV detection. The methods were rapid and a number of commercial samples from a range of suppliers and the results from the CE method showed good agreement with reference methods and with the product specification data. Citrate levels were determined using untreated samples whilst a simple dilution with water was required prior to determination of preservatives. The method therefore offered considerable savings in time and cost compared to the existing methods.

Conclusions

CE offers a number of advantages for the analysis of simple organic acids in a range of analytes which includes speed, reduced cost of analysis and simplicity. The methods are in routine use in a number of industries including foods and pharmaceutical. There are also a number of routine clinical applications.

Kevin Altria is an associate director in the pharmaceutical development department of GlaxoSmithKline. He is editor of "CE Currents" and a member of LCGC Europe's editorial advisory board. Direct correspondence about this column should go to "CE Currents", LCGC Europe, Advanstar Communications, 4A Bridgegate Pavilion, Chester Business Park, Wrexham Road, Chester, CH4 9QH, UK, or e-mail the LCGC Europe editor, Alasdair Matheson, at amatheson@advanstar.com

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(12) http://www.oiv.int/oiv/info/enmethodesinternationalesvin

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