Natalia Belikova, PhD, is analytical services director at SGS Life Sciences, Lincolnshire, IL.

Natalia Belikova

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 reverseâ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. 

	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 

) <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 

. All system suitability requirements were met each day. An example chromatogram from a blank (diluent) is shown in

	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 

	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 

, and the linear regression plot shown in 

	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 

	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 

, with example chromatograms for 50%, 150%, and LOQ spiked samples shown in 

, respectively. Example chromatogram for 100% spiked sample is shown as 

. 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 

; 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 

, 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. 

Validation of Analytical Procedures: Text and Methodology

 (ICH, November 2005.
	2. USP, General Information <1225> 

	When referring to this article, please cite it as N. Belikova, "Determination of Sodium Chloride in Yeast Extract by Ion Chromatography," 

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Articles

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.

Determination of Sodium Chloride in Yeast Extract by Ion Chromatography

Physical, chemical, and biological stability of pharmaceutical molecules is a matter of great concern as it affects the safety and efficacy of the drug product. Stability studies of new drug moiety are mandatory before filing registration. In addition to standard stability studies, forced degradation methodologies of an API can quickly detect if degradants are likely to be generated in a drug substance and help to develop stability indicating methods.

	The quality of an API or finished drug product can be affected over the course of its lifecycle by a range of environmental factors, such as temperature, light, and humidity. But studies to assess any physical, chemical, biological, or microbiological changes that may occur during storage can be complex, time-consuming, and costly.

	Drug substance stability must be considered throughout the drug development process, from the preclinical stage to final product approval. Analytical development, forced degradation studies, and bulk API stability studies form the basis of preclinical work; Phase I studies cover further analytical development and method validation, initial formulation stability studies, and stress tests.

	Test procedures for the assessment of quality levels of pharmaceutical materials are subject to various requirements. In the United States, Section 501 of the Federal Food, Drug, and Cosmetic Act states that assays and specifications 

in monographs of the United States Pharmacopeia 

 Formulary constitute legal standards. The current good manufacturing practice (GMP) regulations [21 

211.194(a)] require that test methods used for assessing compliance of pharmaceutical materials with established specifications must meet proper standards of accuracy and reliability. It is essential, therefore, that proposals for adoption of new or revised compendial analytical procedures are supported by sufficient laboratory data to document their validity.

	GMP regulations-such as FDA guidelines and the International Conference on Harmonization (ICH) Q1A to Q1F guidelines-require a pharmaceutical product to have an expiration date, determined by appropriate stability testing. They also require a written stability study program that specifies sample sizes and test intervals; controlled storage conditions; validated and stability indicating test methods; and that an adequate number of drug production batches are tested. Follow up stability studies of the final product in its marketed container or closure after approval is also required.

	In investigational applications, information must be submitted to support drug substance and drug product stability during preclinical and clinical studies, and the data submitted must be consistent within the period needed.

	With respect to new drug applications (NDAs), submissions for approval by the regulatory authorities are required to contain data from stability studies conducted on both the drug substance and drug product in support of the product expiration date. For abbreviated applications, such as those for new generic drugs, stability data must be included to show that the generic drug has a stability profile comparable to that of the branded products to demonstrate pharmaceutical equivalence.

	Forced degradation studies are a regulatory requirement and scientific necessity during drug development; compared with stability studies, forced degradation studies help in generating degradants in a much shorter time span. The samples generated from forced degradation can be used to develop the stability indicating method that can be applied later for the analysis of samples generated from accelerated and long-term stability studies.

	Forced degradation studies use external stresses to rapidly screen material stabilities by subjecting new drug substances and drug products to conditions more severe than accelerated conditions. These studies are required to demonstrate specificity of stability indicating methods as well as providing an insight into degradation pathways and degradation products of the drug substance while helping to elucidate the structure of the degradation products. Forced degradation studies show the chemical behavior of the molecule, which in turn helps in the development of stable formulations and suitable packaging. Because it is faster and less expensive than conducting longer-term storage tests, the technique is used for rapid selection and elimination tests.

	According to the ICH guideline Q1A(R2), Section 2.1.2) (1), stress testing is intended to identify the likely degradation products, which further helps in determining the intrinsic stability of the molecule and establishing degradation pathways, as well as validating the stability indicating procedures used. Knowledge of the stability of a molecule helps in providing proper storage conditions and shelf life, which are essential for regulatory documentation.

	Forced degradation studies have several aims, including establishing degradation pathways of drug substances and drug products and their mechanisms--such as hydrolysis, oxidation, thermolysis, or photolysis--as well as understanding the chemical properties of drug molecules, and the elucidation of the structure of degradation products. Studies can be used to determine the intrinsic stability of a drug substance in formulation, and to differentiate degradation products that are related to drug products from those that are generated from non-drug product in a formulation; this information can obviously be used to generate more stable formulations.

	That being said, the overriding goal of forced degradation is to produce a degradation profile similar to that which would be observed in a formal stability study under ICH conditions, so that any problems can be brought to light and solved much faster and more efficiently.

	An API was evaluated by a forced degradation study, designed to assess the degradation of the compound when exposed to light, heat (60 °C wet, 105 °C solid), acid (heat/room temperature), base (heat/room temperature), and oxidation (heat/room temperature). The study used five controls:

		Control A-Sample (0.5 mg/mL), no treatment, wrapped in foil

		Control B-Sample (0.5 mg/mL), no treatment, wrapped in foil, placed into the photostability chamber as a temperature control

		Control C-No sample, 5.0 mL of 0.3% hydrogen peroxide, wrapped in foil

		Control D-No sample, 1N hydrochloric acid

		Control E-No sample, 1N sodium hydroxide.

	The photostability testing of drug substances must be evaluated to demonstrate that light exposure does not result in any unacceptable changes. Light stress conditions can induce photo oxidation by free radical mechanism, and functional groups such as carbonyls, nitro aromatic, N-oxide, alkenes, aryl chlorides, weak carbon-hydrogen and oxygen-hydrogen bonds, sulfides, and polyenes are likely to introduce drug photosensitivity.

	Photostability studies are performed to generate primary degradants of drug substances by exposure to ultraviolet or fluorescent conditions. The most commonly accepted wavelength of light is in the range of 300-800 nm to cause the photolytic degradation and the maximum illumination recommended is six million lux hours. 

	The design of photolysis studies is left to the applicant’s discretion, although ICH Q1B (2) specifies that the light source should produce combined visible and ultraviolet (UV, 320-400 nm) outputs, and that exposure levels should be justified.

	To test for light degradation, Control A and Control B were placed into a photostability chamber for light exposure at room temperature. The chamber was set up to fulfill the requirements of ICH Q1B Option 2, which specifies that the same sample should be exposed to both the cool white fluorescent and near ultraviolet lamp. A cool white fluorescent lamp, designed to produce an output similar to that specified in ISO 10977 (1993), and a near-UV fluorescent lamp, having a spectral distribution from 320 nm to 400 nm, with a maximum energy emission between 350 nm and 370 nm were used.

	Standard exposure is specified as 1.2 million lux hours of visible light and 200 watts/square-meter-hours of near-UV at a temperature below 30 °C. The light sources were placed in the top of the chamber and the temperature was monitored during the study using a calibrated environmental monitoring system.

	A 2% aqueous solution of quinine monohydrochloride dehydrate was also exposed during the same period to show that sufficient exposure time had elapsed. The quinine solution was placed into two 10mm quartz cells and sealed, and one cell was wrapped in aluminum foil to act as a temperature control. The length of exposure must be sufficient to ensure a change in absorbance of at least 0.5 between the exposed solution and the temperature control.

	After exposure, the quinine and sample solutions were analyzed within 30 minutes of being removed from the chamber and the actual hours of exposure for each lamp were documented.

	The question of how much degradation is sufficient has been the topic of much discussion among pharmaceutical scientists. Degradation of drug substances between 5% and 20% has been accepted as reasonable for validation of chromatographic assays; however, some pharmaceutical scientists believe that 10% degradation is optimal for use in analytical validation for small pharmaceutical molecules, for which acceptable stability limits of 90% of label claim is common. Others have suggested that drug substances spiked with a mixture of known degradation products can be used to challenge the methods employed for monitoring the stability of a drug product.

	Forced degradation may not necessarily result in a degradation product. The study can be terminated if no degradation is seen after the drug substance or drug product has been exposed to stress conditions (e.g., temperature stress of 60 °C for four weeks). Overstressing a sample may lead to the formation of a secondary degradation product that would not be seen in formal shelf-life stability studies; understressing may not generate sufficient degradation products to develop a selective test method.

	For the API under evaluation, the degradation peak was 3-15% (5-20%). Had this criterion not been met, then the degradation condition could have been adjusted accordingly.

The percentage degradation is calculated relatively to the control, non-stressed sample, by 

	For each condition, the peak shapes from stressed and control samples needed to be equivalent to indicate that no impurities or degradation products co-elute with the active ingredient. Chromatograms for each of the degradation conditions must not have unexpected shoulders or unusual characteristics when compared with the control and the blank. Additionally, for each condition, the peak purity angle of the active ingredient must be less than the purity threshold, demonstrating peak purity. All impurities must be resolved from the main peak with a USP resolution of not less than 1.0, but preferably not less than 1.5.

) can also be used to assess each condition and the following criteria applied depending on the extent of degradation.

When degradation is between 10% and 15%, the lack of mass balance is identified by more than 50% relative deviation (

). For results showing degradation is between 5% and 10%, the lack of mass balance is identified by more than 60% relative deviation, and this changes to 70% relative deviation when degradation is between 2% and 5%. When degradation is less than 2%, results are not amenable to mass balance consideration. However, it should be noted that degradation often results in degradation products that may have different absorbance characteristics, so mass balance criteria cannot always be met.

: Final Degradation Conditions. RT is room temperature.

					 Heat (60 ËC for 24 hours, solution)

					Accelerated Stability 7 Days 40 ËC, 75% RH

	The final degradation conditions and peak purity are shown in 

					Accelerated Stability 7 Days 40 ËC, 75%RH

Forced degradation tests, conducted in early development stages, can provide insight into degradation pathways and products of a drug substance.


Early Efforts Identify Unstable APIs

Forced degradation studies can identify stability problems for drug substance and drug products.