With the ongoing globalization of the world’s food supply, the presence of potential contaminants in food is an ever-important and increasing global safety concern. Developing accurate and reliable screening and confirmatory methods is crucial for successful food safety. With the power of mass spectrometry to provide highly specific qualitative and highly sensitive quantitative data, LC/MS and GC/MS techniques are playing an increasingly important role in the protection and safety of our food supply.
This article describes the benefits of methods using mass spectrometry detection for contaminants testing, and provides some considerations for maximizing chromatographic performance via LC/MS and LC/MS/MS to improve data quality for both food contaminant screening and confirmatory methods. The goal is to provide guidance and insight to assist laboratory analysts in developing reliable and surgical analytical methods that can be used to determine the compliance of food substances with national and international regulations. Depending upon a lab’s goal, different analytical methods and techniques may be combined.
To begin with, a screening method is used to sift through large numbers of samples in a relatively short period of time to find potentially noncompliant samples. The goal is qualitative in nature and meant to determine if any of these contaminants are present in this sample.
Ideally, all the samples will turn up negative, but if some positive indications of contamination result from the screening method, a confirmation step is necessary to positively identify those suspect contaminants using a very selective technique such as LC/MS. This confirmatory second method will definitively identify which samples are contaminated, and to what levels. Thus, both qualitative and quantitative results are required.
MS detection has greatly improved the ability to positively identify suspected contaminants at extremely low levels in samples, and this has resulted in LC/MS techniques becoming important and reliable analytical tools. Simultaneously, mass spectrometry can provide valuable information, including:
- The chromatographic retention time, which can be indicative of a contaminant’s chemical nature
- The mass-to-charge ratio for an ion—a function of its molecular composition and structure
- The multiple reaction monitoring (MRM) ratios, which help identify a substanc
- A mass spectrum for an ion—essentially a unique chemical fingerprint for a given molecule.
Of course, in addition to all of these qualitative data, which help to positively identify a suspect contaminant, quantitative data can tell us how much is present in a sample.
Depending on the samples of interest, screening methods can be created with liquid chromatography/ultraviolet (LC/UV) or gas chromatography/flame ionization detection (GC/FID), as long as the developed methods are robust and selective enough. However, when it comes to confirmatory methods, an LC/MS method is the “gold standard” because it will provide the crucial information about the compound structure.
Per the European Union (EU), confirmatory methods are designed to provide full or complementary information, enabling the substance to be unequivocally identified and quantified to a specific level, if necessary. A few considerations to be reviewed are:
Figure 1 – Equation showing how chromatographic resolution (Rs) is affected by column efficiency, selectivity, and capacity.
- The minimum retention time of target analytes should be at least two times the void volume of the column. The primary reason for this is that the early portion of a chromatogram often contains regions where poorly retained components of the sample matrix elute. This is the root cause of matrix interference and often affects accurate quantitation. Thus, it is important to make sure that the target analytes are far away from the early portion of the chromatogram, if at all possible, to help ensure accurate quantitation.
- The retention of a target analyte should match the retention of a matrix-matched calibration standard. This is good chromatographic and lab practice.
- The ratio of the retention of the target analyte to its specified internal standard should be constant within an acceptable range. This helps ensure that the chromatographic method is robust and repeatable, giving consistent retention times from run to run.
For the analysis itself, the success of any given chromatographic method depends on a number of factors, including:
- The column length, particle size, and stationary phase.
- The mobile phase—the nature of both the aqueous portion and the organic component. Different solvent combinations will ultimately affect the selectivity of the method.
- The running conditions—whether isocratic or gradient is used along with the type of solvent profile.
Important in every chromatographic method is resolution or separation of the key compounds of interest. Figure 1 is an equation that shows how chromatographic resolution (Rs) is influenced by three main factors— column efficiency, selectivity, and capacity. The relative contribution of each of these three factors to resolution is shown in Figure 2, and one can begin to see different ways in which separations can be affected. For instance, k is very important. If analytes are not retained, they cannot be separated, but too much retention will potentially cause peaks to become too broad; thus the capacity factor is not effective after a certain point.
Figure 2 – Contribution of column efficiency, selectivity, and capacity to resolution.
In contrast, as efficiency is increased with options such as core-shell media, a rapid and continuing improvement in resolution is seen. This means that as column efficiency is increased, there is potential to improve resolution.
The third important factor is selectivity, which has the largest influence on the resolution of a separation. However, selectivity is also the most complex factor because it is a function of both the mobile phase and the column stationary phase. This is why adjusting the mobile phase or moving from one column type to another can often have such a dramatic effect on chromatographic results. It is also why it is very important to have both complementary and orthogonal stationary phases as well as various solid supports available for initial method development.
In general, there are three main types of solid supports (Figure 3) that offer the necessary range of selectivities for success in the analysis of food samples by HPLC.
Figure 3 – Types of HPLC media supports.
Conventional fully porous silica
Conventional fully porous silica is general silica media that is available in a broad range of stationary phases and particle sizes and is a good, all-around particle for chromatography. It is stable up to about a pH of 8.
Core-shell particles are the newest advancement in chromatographic media and consist of a solid, impermeable inner core that is surrounded by a thin outer layer of fully porous silica. This unique morphology minimizes all the sources of band broadening and delivers efficiency values that are much greater than with columns packed with conventional fully porous particles. Core-shell technology enables very high separation efficiencies, narrow peaks, increased productivity, and improved resolution.
Organosilica particles are unique in that part of the silica backbone has been replaced by carbon to greatly increase pH stability. Unlike traditional fully porous media, organosilica particles can withstand pH values greater than 10. This can be a valuable tool if the sample is not stable or if an improvement in MS response in negative ion mode is desired by using an alkaline buffer.
HPLC stationary phases
Within each of the solid support categories, there are a few HPLC stationary phase classes. The standard alkyl-bonded phases, the most common of which is C18, can be used as a starting point for a majority of applications. They offer excellent hydrophobic-focused selectivity. The phenyl phases, which consist of a phenyl ring attached to the underlying silica by a carbon linker of varying length, offer an alternative selectivity with both aromatic and hydrophobic functionality. For more balanced polar retention, polar-embedded and polar-endcapped phases can be used. The polar-embedded phases incorporate a polar functional group into the alkyl chain itself. Meanwhile, polar-endcapped phases replace traditional trimethyl hydrophobic endcapping with a small, polar function group. In both of these cases, the incorporation of polar groups alters column selectivity enough from the original alkyl or phenyl phases to enhance selectivity for polar analytes and create stability under highly aqueous conditions.
Despite the wide variety of phases available for the testing of food contaminants, about 80% of the columns sold for reversed-phase separations are C18. In reversed-phase mode, where separation of molecules is based primarily on differences in hydrophobicity, C18 phases have the greatest potential for success. Historically speaking, since C18 phases work most of the time for most samples, logically this is a good place to start. For contaminant analysis of widely varying functionalities, however, considerations in media supports and stationary phase classes definitely help.
Method development with complex samples for contaminants analysis may be a daunting task. However, taking into consideration some fundamental HPLC method development principles and guidelines can greatly improve both the analysis results and the method development experience.
With the power of mass spectrometry combined with developments in LC column technology, such as Kinetex Core-Shell Technology (Phenomenex, Torrance, CA), the food industry will continue to develop more reliable and sensitive methods. With the ever-changing requirements of global food testing regulatory organizations, these improvements can significantly support efforts to ensure that what we consume is safe.
The authors are with Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501-1430, U.S.A.; tel.: 310-212-0555; fax: 310-328-7768; e-mail: [email protected] phenomenex.com ; www.phenomenex.com