Greg Martin, Tran Pham and Markus Wanninger

Waters Corporation, Milford, MA, USA

 


INTRODUCTION

Controlling risks that compromise analytical method performance is an important aspect of Method Lifecycle Management (MLCM). MLCM is an approach to method management that ensures methods are fit-for-purpose throughout their lifetime. Assessing the risks and implementing control strategies are part of applying Analytical Quality by Design (AQbD), a risk-based approach to method development1 and is an integral part of MLCM, Stage 1: Method Design and Development.

 

Typical method-related risks associated with vial quality include adsorption of analyte, appearances of unexpected or "ghost" peaks, ionic effects such as ion suppression or pH changes, mechanical effects (e.g., a septa blocking the autosampler needle), and evaporation and chemical incompatibility caused by interactions between the septa/caps and sample.

 

Understanding the method performance objectives as outlined in the Analytical Target Profile (ATP) and determining the correct vial for the intended method in MLCM Stage 1 can significantly improve the quality, reliability, and consistency of data generated by the analytical method. Controlling the vials used during sample handling and analysis also reduces aberrant data investigations and helps maintain good method performance.1

 

The case studies in this paper illustrate the types of issues that occur when using poor quality vials. These case studies prove that the root cause for poor data quality is poor quality vials. Methods for choosing the appropriate vial type to control the risks of these issues from occurring are also discussed. This paper focuses on the use of glass vials for LC and LC/MS small molecule applications. To address challenges of data quality involving similar applications for large molecules such as peptides and proteins, please reference Waters white paper, "Achieving Maximum Protein and Peptide Recovery, Sensitivity, and Reproducibility using Quan Recovery Vials and Plates" (p/n: 720006571EN).

 


CASE STUDIES

LC/GC

Unexpected or Ghost Peaks

One of the potential risks of HPLC testing is the presence of unexpected or ghost peaks resulting from the HPLC vials. Such peaks may be due to contaminants from the vial, septa, or the unpredictable degradation of the analytes caused by impurities present in the glass.

 

Degradant peaks induced by the alkaline impurities from the glass vials were detected in two ezetimibe solutions.2 Each solution was stored in two different vials (Figure 1). To prevent the appearance of unexpected peaks during LC/UV analysis, Waters™ LC/GC Certified Vials may be used as a control strategy. Waters LC/GC Certified Vials have appropriate manufacturing and quality control procedures to ensure these glass vials do not contain impurities that can compromise data quality.

Figure 1 Normal and Ghost Peaks

Figure 1. Top chromatograph shows normal behavior, and the bottom chromatogram shows ghost peaks

 

LC/MS

Impact of glass cleanliness and purity3

Sensitivity is important when analyzing samples via LC/MS and when the presence of background noise prevents the ability to accurately detect and quantify peaks. Background noise can come from many factors such as instrument, sample, and vial cleanliness.

 

Variability in the levels of vial cleanliness from one manufacturer to another can be seen in the MS scans of a solvent sample stored in a competitor's vial and in a Waters LCMS Certified Vial (Figure 2). The solvent samples were stored in the vials for a fixed amount of time, removed, and analyzed via LC/MS. The competitor's vial scan is typical of vials commonly purchased around the world. The reference scan is from a solvent sample that was not stored in a vial.

 

To mitigate the presence of background noise during LC/MS analysis, Waters LCMS Certified Vials may be used as a control strategy to ensure peak detection and quantitation are accurate to further enhance data quality.

Figure 2 Waters and Competitors MS Scans of Vials

Figure 2. Comparing typical MS scans of Waters LCMS Certified Vials, vials from other sources, and clean solvents.3

Besides the cleanliness of a glass vial, the glass composition can also affect detection sensitivity. In the case study below, it was found that the analyte sofosbuvir showed a measurable level of degradation while sitting in the sample vial (Figure 3}. This degradation presented itself as a second peak eluting just before the main component. This initiated a study
that demonstrated the glass composition and the sample reacting with the glass components were the root causes of degradation. It was clearly shown that this effect varied greatly depending on the source of the glass (Figure 4).3,4

Figure 3 24hr chromatogram showing degradation peak

Figure 3. Overlay of 24 h sample chromatogram of sovosbuvir showing degradation peak caused by interaction of sample with the glass vial.4

 

Figure 4 Graph of empimerization over time

Figure 4. Graph of empimerization over time, showing the normalized area % of epimer. The monitored vials A (Waters) and 8-0 show different levels of degradation.4 

 

LC/MS

Impact of glass cleanliness and purity3

In quantitative or low concentration qualitative LC/MS analysis, loss of analyte due to non-specific adsorption, non-specific binding on the surface of glass vials and a
high level of vial-to-vial variability are detrimental to data quality.5,6 This is a significant problem in the lab because such interactions are often not recognized early enough and can happen immediately or over the course of a few hours while the sample is waiting for injection. The result is the cost of lost development time and cost of troubleshooting time. The extent of lost analyte has been shown to vary significantly among vials from different manufacturers
(Figure 5). Waters TruView™ LCMS Certified Vials showed the lowest adsorptive losses and consistently performed better than competitor vials (Figure 6). Vial-to-vial adsorption variability demonstrates the variability between vials within a package.

Figure 5 Comparison of adsorptive loss of chlorhexidine acetateFigure 5. Comparison of adsorptive loss of chlorhexidine acetate in a variety of vials.

 

Figure 6 Vial-to-vial adsorption variability within a pack

Figure 6. Comparing vial-to-via/ variability (%RSD) for adsorptive loss of chlorhexidine acetate in vials from different vendors.

 

The impact of vial source variability on absorption is shown in Figure 7 where it can be seen that the nortriptyline peak is much smaller after four hours in vials from Vendor A and Vendor B. It is essentially unchanged in Waters TruView LCMS Certified Vials.

Figure 7 Impact of adsorptive loss of nortriptyline in several vials

Figure 7. Impact of adsorptive loss of nortriptyline at 7 ppb concentration in several vials. The tallest peak represents the initial injection, the smaller peak (overlaid) is the injection after four hours.6

 


EXTRACTABLES FROM CAPS AND SEPTA

In addition, the glass, caps, septa, and even the packaging components of the vial can affect the chromatograms and data quality. This is especially true when working with low concentrations of analytes and using mass spectrometry. To demonstrate this, 1 ml of 95% methanol in water was added to vials from different sources. The caps were attached and the vials were incubated at room temperature for one hour. Septa were punctured three times with an autosampler and the solutions were analyzed by LC/MS. The results, shown in Figure 8, show that the TruView LCMS Certified Vials showed mass spectra like that of the solvent blank, but numerous peaks, at various masses, were observed with vials from other sources. 

Figure 8 Comparison of extractables from septa in different vials

Figure 8. Comparison of extractables from septa in different manufactured vials and the TruView LCMS Certified Vials.

 

The previous case studies demonstrate that selecting vials that are not fit-for-purpose with current method development can lead to costly and time consuming investigations. To avoid unexpected delays, implement risk assessments as recommended in Stage 1 of MLCM in order to fully assess different vials and how they affect data quality. The following section describes the different types of vials and provides guidance in choosing the most suitable vial for your method's purpose.

 


VIALS FOR CHROMATOGRAPHIC USE

There are two main components to a sample vial - the glass vial and the cap with septa. Each of these components can have a potential impact on the analytes of interest and should be considered when choosing a vial for robust method development.

 

A typical sample vial is made of USP Type 1 Borosilicate glass which meets a certain level of USP requirements. It is important to remember that glass is an inorganic compound which exhibits an active surface and contains various levels of metals and free ions that can interact with the stored analytes. These metals and free ions are not considered by the USP guidelines. This means that the same USP glass type in sample vials from various manufacturers can cause variations in analytical results since the surface activity is not entirely specified.2

 

During the typical residence time of a sample solution in a vial, it is possible for glass components such as sodium and other alkali metals to leach into the solution. This can significantly raise the sample pH and trigger changes such as unexpected degradation and lower analyte recovery. Other ions blooming to the glass surface can cause ion suppression effects in the ESI source of the MS and therefore alter the results. The polar binding of the analyte to the glass surface such as non-specific adsorption (NSA) or non-specific binding (NSB) can adversely affect detection or quantification. Analyte binding can be partially mitigated by surface alteration2, 5 during manufacturing.

 

The second component is the caps and septa. In addition to the common considerations of chemical compatibility (Table 1), the cleanliness of the septa is important. During the manufacturing process, the septa material undergoes several finishing and conditioning processes that define the final purity of the septa. Impurities from the septa can be introduced into the sample vial by outgassing or contamination at the point of sample injection.

 

Table 1. Overview of solvent compatibility and suitability of different septa materials
Table 1 Overview of solvent compatibility and suitability of different septa materials

 

Contaminants can originate from inside the glass vial, septa, and packaging. Polymers are commonly released by the septa. Contaminants from inside the sample vial are typically generated from lubricants and oils from the machines used during glass handling processes. Antistatics and other volatiles are known contaminants from packaging materials. Some level of these contaminants can be controlled by the manufacturing process, but it is important that the level of contamination does not exceed the sample and detection requirements of the analysis.

 

Glass vials, caps, and septa quality should be considered when assessing method-related risks to achieve robust methods and good quality data. Choosing a manufacturer that can produce high quality vials with little to no lot-to-lot variation can serve as a control strategy to minimize such risks. The Waters Certified glass product line discussed in this paper offer a variety of fit-for-purpose, high quality, and dependable glass vials. This helps ensure methods can generate good quality data, ultimately achieve good method performance, and support the QbD method development approach as suggested by MLCM.

 

LC/GC CERTIFIED VIALS

LCGC Certified vials

Waters LC/GC Certified Vials are fit-for-purpose for LC/UV analysis and tested for contaminants that can effect results at the parts per million (ppm) or µg/mL level.

 

The certification test of the vial is based on characterization of common chemicals used during manufacturing and contaminants introduced from typical packaging materials. The LC/UV certification test is performed after the vial has spent at least four days inside its packaging. This ensures that pH changes for an aqueous solution are within the specifications. Additionally the septa material is tested with GC headspace to ensure the appropriate finish of the septa materials.

 

LCMS CERTIFIED VIALS

LCMS Certified vialsWaters LCMS Certified Vials are suitable for routine LC/MS applications, such as those that use the ACQUITY"' QDa"' or other single quad mass spectrometers and for analyte concentrations in the 10's to 100's ng/mL (parts per billion, ppb).

 

The LC/MS certification involves testing for contaminants typically found in lower end quality vials that can interfere with the electro spray ionization, such as surfactants, lubricants and antistatic agents and silicone polymers from septa.

 

TruView LCMS CERTIFIED VIALS

TruView LCMS certified vialsTruView LCMS Certified Vials are best suited when a high analyte recovery is required with analyte concentration levels of 1 pg/ml or below (1 ppb or below). The glass surface of these vials exhibits very low adsorption and is ideal for storing solutions that have polar analytes at low level concentrations. The low adsorption is achieved by following tightly controlled manufacturing process conditions. In addition to the MS cleanliness tests, TruView LCMS Certified Vials are certified using UPLC"'/MS/MS (MRM) for low analyte adsorption.

 

Every application requires different types of vials and therefore it is recommended to determine the best vial type that satisfies the application's needs and requirements.

How to choose what type of vial is most appropriate for your application:

  • If you require sensitivity in the low ppb range or below, TruView will give you the best performance.
  • If you will use MS for detection for a routine application with concentration in the low- to mid-ppb range, choose LCMS.
  • For routine analysis, such as HPLC with UV detection or GC, and typical sample concentrations in the ppm range, LC/GC certified vials will meet your needs in the most cost-effective manner.

Table 2. Testing levels for Waters Certified Vials

Table 2 Testing levels for Waters certified vials

 


CONCLUSIONS

It has been shown that vial quality does have an impact on data quality and should be considered when assessing method-related risks during Stage 1 of MLCM: Method Design and Development. Choosing the right vial, cap and septa for sample analysis is important
to ultimately achieve and maintain good method performance and is essential for method robustness. The sample vial coming from a single, specified source also reduces risks when the sample methods needs to be deployed to different laboratories across the globe.

  • For non-MS applications and sample concentration ≥pg/ml, Waters LC/GC Certified Vials are a good choice. Adsorption would have little effect on reported results due to high analyte concentration and low glass surface area.
  • For LC/MS applications, with sample concentrations in the 10-100's ng/ml, Waters LCMS Certified Vials are a good choice. These vials have fewer masses seen by MS and high analyte concentration would have less effect on results.
  • For MS applications with sample concentration in the ng/ml and lower range, Waters TruView LCMS Certified Vials are the best selection. This vial combines low adsorption and few masses seen by MS.

Waters sample vials offer the reliability and reproducibility required for consistent analytical method performance and reduce aberrant data investigations.

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References

  1. <https: //www. la bcom pa re.com/10-Featu red-Articles/360458-Future-Proofing-Pha rmaceuticaI­ Analytical-Method-Development-With-a n-AQbD-Approach/?a rev=true>

  2. Jin, J.; et al. Ghost Peaks of Ezetimibe: Solution Degradation Products of Ezetimibe in Acetonitrile Induced by Alkaline Impurities from Glass HPLC Vials. Journal of Pharmaceutical and Biomedical Analysis. 2017, 740, 281-286.

  3. Waters LCMS Certified Sample Vials, White Paper-Waters Corporation, 720001517EN, 2008.

  4. Arvary, M. The Importance of Vial Composition in HPLC Analysis: An Unusual Case of Phosphorous Pseudorotation. Journal of Pharmaceutical Biomedical Analysis. 2017, 734, 237-242

  5. Achieving Maximum Protein and Peptide Recovery, Sensitivity, and Reproducibility using Quan Recovery Vials and Plates. White Paper-Waters Corporation, 720006571EN, 2019.

  6. Waters TruView LCMS Certified Sample Vials. White Paper-Waters Corporation, 720004097EN, 2011.