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Use of Monolithic HPLC Columns for Food Analysis in Complex Matrices

Mon, 12/23/2013 - 8:00am
Dr. Egidijus Machtejevas, Product Management Analytical Chromatography and Dr. Stephan Altmaier, Research and Development Analytical Chromatography, Merck Millipore

Chemical analysis of food is a prerequisite for ensuring correct labeling and protection of consumers against adulteration and misbranding. Nutritional data on packaged food are necessary for helping consumers choose food in accordance to their individual dietary needs.  Accordingly, labeling regulations describe in detail the requirements for nutritional labeling (nutrients, amounts and caloric values) on food packages. To ensure a consistent nutrient declaration, the food manufacturer needs to perform additional testing for nutrients such as sugars, organic acids, sugar alcohols, fat and fatty acids, protein, and sodium, as well as for vitamins and minerals.

Measures for ensuring food safety, provision of wholesome food, and consumer protection against adulteration and misbranding require reliable data obtained by chemical analysis of food. Reliable analytical results are also essential to facilitate international food trade. In an attempt to provide a safer food supply, the FDA recently unveiled new, more stringent rules on imported foods. Under the new standards, importers must know whether supplying farms or processors are taking steps to eliminate risk.

In food analysis, complexity of the food matrix can have the largest impact on the performance and long term reliability of the analytical methods and procedures used. The food matrix consists mainly of chemical compounds including protein, carbohydrate and lipids, which can significantly affect the performance of analytical methods. For example, high-fat or high-sugar foods can cause different types of interferences compared to low-fat or low-sugar food [1].

Application of different sample preparation procedures, being a pre-condition for delivery of accurate analytical results, is often not only time-consuming, but sometimes leads to artifact formation as most of the errors come from sample preparation [2]. Therefore, analytical methods for food analysis always need to take into account the characteristics and composition of the specific food matrix which in general shorten the life time of the analytical column (in extreme cases only, one injection and the column is dead).

As such, there is an increasing need for new analytical methods able to cope with analytes in very complex matrices. These new analytical assays must provide sensitivity, robustness and high resolution within an acceptable analysis time. Many modern approaches in high pressure liquid chromatography (HPLC) analysis enable the reduction of the analysis time without compromise on resolution and/or separation efficiency including the use of monolith HPLC columns.

Monolithic columns are made of a single rod of high purity porous silica and are substantially more matrix tolerant than particle packed columns of similar efficiency. The high permeability and porosity of the rigid silica skeleton result in low column backpressure and more flexibility in flow rates compared to particulate columns. Column equilibration after gradient elution is also much faster on monolithic columns than similar dimension particle packed columns. Overall this offers a possibility to perform high throughput analysis without losing separation efficiency or peak capacity.

Following, we provide examples of how monolithic columns can be used to accelerate time to results and help overcome the inherent challenges presented by analyzing molecules of interest in very complex matrices.

Carbohydrate Analysis

Low molecular weight carbohydrates, the monosaccharides and disaccharides, are commonly referred to as sugars. Beside monosaccharides and disaccharides, there are also neutral sugars, acidic sugars, amino sugars, sugar alcohols and their various isomers. Low molecular weight saccharides are common in food, such as fruits, honey and sweets. The separation and identification of saccharides is challenging, especially for compounds having the same chemical formula and only small differences in their molecular structure, i.e. disaccharides maltose and isomaltose. In addition, carbohydrates from simple sugars to oligo- and polysaccharides represent a detection challenge in that they are lacking chromophores.

Various separation techniques have been used for carbohydrate analysis including anion chromatography, size exclusion chromatography, reversed phase chromatography, hydrophilic interaction liquid chromatography (HILIC), GC and TLC. HPLC using aminopropyl functionalized columns is one of the more common techniques for analysis of saccharides. In this technique, a mixture of acetonitrile and water is used as the mobile phase and the retention increases with reduction of water content just as in HILIC. In addition, the higher the molecular weight of the sugar, the longer it takes to elute. The aldehyde radicals in sugars can react with the amino radicals in the stationary phase to create Schiff bases, which can sometimes cause significant tailing on peaks for pentasaccharides (such as arabinose and ribose). This can be inhibited by adding a salt to the mobile phase.

An advantage with an amino column is that it catalyzes the mutarotation of reducing sugars effectively causing the retention time of the sugar to be the average of its two anomers, showing as only one peak in the chromatogram. Figure 1 illustrates how Chromolith® NH2 columns can be used for more effective separation of monosaccharides, disaccharides and glycoalkaloids (alkaloids with sugar moieties).

Figure 1.  Separation of monosaccharides (A), disaccharides (B), and glycoalkaloids (C) using Chromolith® NH2 columns.

  

Colorants

Food additives such as colorants are intentionally added to food and must be safe for a lifetime of consumption based on current toxicological evaluation. Food colorant is any dye, pigment or substance that imparts color when it is added to food or drink. Colorants are used in foods for many reasons including:

  • Offset color loss due to exposure to light, air, temperature extremes, moisture and storage conditions
  • Correct natural variations in color
  • Enhance colors that occur naturally
  • Provide color to colorless and "fun" foods

Food colorings are tested for safety by various regulatory bodies. In the United States, FDC numbers are given to approved synthetic food, while E numbers are used within the European Union for all additives, both synthetic and natural, that are approved in food applications.

Some artificial colorants are only allowed to be used for limited use and others are banned or delisted. For instance Orange B (red shade) is allowed only for use in hot dog and sausage casings, whereas FDC Red No. 2 (Amaranth) is banned for use in food in the United States.

Figure 2 shows the high resolution, rapid separation of a variety of artificial food colorants including Amaranth using a Chromolith® High Resolution RP-18 column.

Figure 2.  Detection of artificial food colorants using Chromolith® High Resolution RP-18 column.

Flavonoids and Isoflavones

Flavonoids are a class of plant secondary metabolites. Flavonoids are all ketone-containing compounds and the IUPAC nomenclature classify flavonoids as:

  • Flavones from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure (examples: quercetin, rutin) 
  • Isoflavonoids from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure
  • Neoflavonoids from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure

Food manufacturers have become interested in flavonoids for their possible medicinal properties.  Although no physiological evidence is yet established, the beneficial effects of fruits, vegetables, tea and red wine have sometimes been attributed to flavonoid compounds. Sources of flavonoids include all citrus fruits, berries, ginkgo biloba, onions, parsley, pulses, tea (especially white and green), red wine, seabuckthorn and dark chocolate.

When analyzing flavonoids in fruit, vegetables and other flavonoid-rich solid samples, problems may arise from the sample matrix. The analysis of flavonoids thus constitute problems with finding suitable sample preparation, to resolve all interesting compounds from each other chromatographically, and to detect them accordingly. 

Figure 3.  Analysis of flavonoids (A) and isoflavonoids (B) using Chromolith® FastGradient RP-18 columns.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3 illustrates use of Chromolith® column, combined with MS detection, to develop sensitive methods for monitoring flavonoids and isoflavones in food and beverage samples.

Food safety and consumer protection are increasingly important and require new analytical techniques.  Incorporation of monolithic silica columns into food analysis procedures can help address challenges presented by complex matrices.  These columns can be used with any HPLC instrument and detection technique, i.e. UV, fluorescence (FL), refractive index (RI), electrochemical (EC), evaporative light scattering (ELSD), and mass spectrometric (MS) detection and are available in a wide range of sizes to allow easy scaling of methods.

REFERENCES

[1]        “Introduction to food analysis” Nielsen, S.S. in: “Food Analysis, Springer US, USA, 4th Edition, 2010, 5-13.

[2]        “Quantitative determination of beta-carotene stereoisomers in carrot juices and vitamin supplemented (ATBC) drinks” Marx, M. et al. Food Chemistry, 70, 2000, 403-408. “United States Government Regulations and Standards”, Nielssen, S.S. in “Food Analysis, Springer US, USA, 4th Edition, 2010, 15-33.

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