Determination of Sodium Chloride in Yeast Extract by Ion Chromatography

March 2, 2019
Natalia Belikova
Pharmaceutical Technology Europe
Volume 2019 Supplement, Issue 1
Page Number: s16-s20

Determination of sodium chloride level is critical for assessment of purity of yeast extracts. This case study demonstrates the validation of an ion chromatography method as a suitable analytical approach.

Yeast extract is commonly used as an excipient in cell culture, providing a range of nutrients that the cells require to grow. However, as a raw material it may contain variable levels of sodium chloride (NaCl), depending on the purification process. That is a problem, as NaCl concentration must be carefully controlled during the fermentation process; therefore, osmotic pressure in the media will not vary and cell growth is not to be inhibited.

The process of assessing salt levels in yeast extract can be challenging because of the complex nature of the mixture. Traditionally, titration-based methods are used to quantify exact sodium or chloride levels in inorganic salts, but in this case, they are unsuitable. There are several reasons for this, including the fact that the salt level might be too low to be determined accurately, and because the yeast extract itself can interfere with the reaction components, and therefore affect the colour reaction. Furthermore, sample sizes required for titration may be fairly large. While yeast extract is relatively inexpensive, smaller sample sizes are always preferred.

A far more suitable option for measuring low salt levels in complex biological samples is to use ion chromatography. Salts usually readily dissolve in aqueous media, giving free cations and anions that can be measured with good accuracy, precision, and specificity via ion chromatography. Other methods, such as those based on the colour reaction for identification and ion-specific electrode titration, can be used for pure NaCl samples, but they are not suitable for quantifying residual salt in complex biological matrices, and therefore are not applicable in this case.

For a successful analysis, the sample should be readily soluble in water in order that all the ions are available for liquid chromatography separation and detection. If the sample is not completely soluble, a small amount of organic solvent can be used to promote dissolution, but this should be minimized where possible for ion chromatography. In contrast to reverse‑phase chromatography, ion chromatography separation is based on the column’s affinity to specific cations and anions. Ion exchange columns should not be used when high levels of organic solvents are present.

Another potential problem arises from the fact that the sample material should be able to pass through the column without killing it. The method should be sufficiently robust that the column remains functional for multiple sample injections. 

Case study

In this example, the client specification for NaCl was no more than 5.0% w/w of the sample. As it is just one component of the complex yeast extract matrix, traditional titration methods were clearly inappropriate, and so a method based on ion chromatography was developed instead. 

The technique uses a Model ICS-2100 ion chromatography system (Dionex), equipped with a conductivity detector to separate and quantitate the level of sodium ions present in the yeast extract sample. It also has an injection valve, pump, guard column, column, chemical suppressor, and data collection system. The amount of NaCl in the sample was calculated based on a 1:1 stoichiometry for sodium and chloride ions, and the assumption that salt was the only source of free sodium ions within the sample.


The separation was performed on a IonPac CS12 250×4 mm column (Dionex), with an IonPac GC12 50×4 mm guard column (Dionex), and using a flow rate of 1.0 mL/min of 10 mM methanesulfonic acid (MSA), generated by the system online using reagent-free ion chromatography (RFIC) technology. The injection volume was 25 µL; both the column temperature and the conductivity detector temperature were 35 °C. A 4 mm suppressor with 30 mA suppressor current were used. A sample size of 1 mg/mL of yeast extract was injected. Sample concentration was low enough to promote sample solubility, but sufficient enough to detect sodium ions with acceptable accuracy and precision.

The validation design was based on International Council for Harmonization ICH Q2B (1) and United States Pharmacopeia (USP) <1225> (2) recommendations. The validation study is protocol-driven, and typically includes testing of system suitability, specificity, linearity, limit of quantitation, precision, intermediate precision, and accuracy. Robustness of chromatographic conditions and solution stability give information about the method’s limitations, and give additional information if troubleshooting of the method will be required for future use.

Method accuracy was evaluated on samples spiked at the limit of quantitation of 0.1% NaCl, 50% of the specification level (i.e., 2.5% NaCl, 100% of the specification level of 5.0%, and also 150% of specification, 7.5%). The recovery of sodium at the limit of quantitation level was within 67–106%, and at other levels was 93–105%. The stability of the standard and the sample was evaluated after three days at both refrigerated conditions of 2–8 °C and at room temperature. 

System suitability was checked before each validation run, and validation solutions were not evaluated unless the system suitability criteria were met. This was done by first injecting the diluent, USP High Quality Water (E-pure), and then the working standard six times to check system precision. Each validation solution was then injected once, with bracketing every six validation injections with a working standard injection, and at the end of the run. Results are shown in Table I. All system suitability requirements were met each day. An example chromatogram from a blank (diluent) is shown in Figure 1 and from a working standard in Figure 2.

Specificity and selectivity were then checked, using injections of USP High Purity Water (diluent), working standard, unspiked sample solution, and sample solution spiked at the 100% concentration level of sodium. These were evaluated for chromatographic interference and resolution from other peaks. No interference from the diluent was observed. The resolution between the sodium peak and the nearest peak in the sample solution was 2.6, whereas in the accuracy 100% spiked sample, it was 2.8, and specificity criteria were met. Example chromatograms for the sample and spiked sample are shown in Figures 3 and 4.


Linearity was assessed by injections of five standard solutions prepared at concentration levels ranging from 0.1% to 7.5% NaCl. Each standard was injected once, and linearity determined through linear regression using the peak areas of sodium peak. The correlation coefficient was 0.99999, and the acceptance criterion was met. The results are summarized in Table II, and the linear regression plot shown in Figure 5.

The limit of quantitation (LOQ) was determined by injecting the linearity standard at lowest level six times. The signal-to-noise ratio calculated for the sodium peak was 75.6; the limit of quantitation has been established as 1 µg/mL, or 0.1% NaCl in regard to the sample amount (1 mg/mL). An example chromatogram is shown in Figure 6.

Accuracy was determined using a yeast extract sample that was spiked with sodium across the LOQ-150% range from the target concentration. The samples were prepared in triplicate at LOQ, 50% and 150% levels, and six samples at the 100% level. An unspiked sample was used as a control to determine the endogenous amount of sodium present in the yeast extract. The percentage recovery at each concentration level was determined, with recovery calculations based on the spike contribution alone. The average level of sodium in the unspiked samples was subtracted from the spiked ones. Results are summarized in Table III, with example chromatograms for 50%, 150%, and LOQ spiked samples shown in Figures 7(a), (b), and (c), respectively. Example chromatogram for 100% spiked sample is shown as Figure 4. Acceptance criteria for accuracy stated in the validation protocol (50–150% for LOQ level and 80–120% for other levels) were met.


Precision was determined using individually prepared yeast extract samples spiked at the 100% level concentration. Six of these samples were prepared and run. The percentage recovery was determined for each spiked sample preparation using the approach that was described in the accuracy section. Precision was calculated based on percentage recovery. The results from the accuracy test on comparable samples were used for this evaluation. Sodium chloride levels were less than 5.0% in the unspiked solution, and the results are summarized in Table IV; the acceptance criterion for precision (% RSD for % Recovery NMT 5.0%) was met.

Similarly, intermediate precision was assessed, with a second analyst repeating the precision test on a different day using a different column and guard column on the same ion chromatography system; these results are summarized in Table V, and, again, the acceptance criteria were met.

In conclusion, the test method for the determination of NaCl in yeast extract by ion chromatography was successfully validated. The method is specific, linear, precise, and accurate, with a limit of quantitation of 0.1% of sodium chloride in the sample. Instrument parameters and sample preparation can be used as a starting point for method development/validation for other requests. 


1. ICH, Q2(R1) Validation of Analytical Procedures: Text and Methodology (ICH, November 2005.
2. USP, General Information <1225>

1, USP-NF.

Article Details

Pharmaceutical Technology Europe
Supplement: Outsourcing Resources
March 2019
Pages s16–s20


When referring to this article, please cite it as N. Belikova, "Determination of Sodium Chloride in Yeast Extract by Ion Chromatography," Pharmaceutical Technology Europe’s Outsourcing Resources Supplement (March 2019).

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