Just juice?

With the constant focus on the health impact of beverages we consume, laboratories need to accurately characterise carbohydrates in fruit juices within record time

SOFT drinks, fruit juices, vegetable juices, formulated juice drinks and other prepared beverages are popular thirst quenchers that are carefully marketed and formulated to provide brand recognition, unique flavour and reliable product stability. Fruit juices contain vitamins, natural antioxidants, minerals and other valuable constituents, in addition to natural sugars. The apparent health aspects of these products, both for growing children and increasingly obese adult populations, has led to widespread criticism of soft drinks and fruit drinks that are instead formulated with a broad range of artificial or modified natural ingredients and are alleged to be far less beneficial in the diet than natural fruit juices.

Food Quality Control (QC) laboratories are constantly challenged to meet the demand for fast and accurate results for the wide range of product constituents found in juice and beverage products. The greatest opportunities in beverage labs exists with the small molecule separations of naturally occurring components and artificial additives, including fortified and natural vitamins, flavours, colourants, preservatives, non-nutritive sweeteners, natural sugars, and high fructose corn syrup for example. High-performance liquid chromatography (HPLC) is an analysis technique that combines a separation mechanism with varied detection strategies for “seeing” what is in a sample and for quantifying the amounts of identified compounds. When we think of fruit juices and other beverages, one of the essential measurements is the carbohydrate content, which is then used to generate or support a caloric claim on the product label.

This article describes the quantitative and qualitative analysis of carbohydrates in fruit juices and juice beverages using HPLC. Simple carbohydrate separations can be performed on functionalised silica or resin-based columns. Refractive index detection (RID), the historical standard for HPLC carbohydrate detection, can be unsuitable because of instability to temperature and mobile phase fluctuations and further because it is not compatible with gradient elution chromatography. Although most carbohydrate separations are isocratic, gradient elution with varying solvent composition is highly desirable for diverse sample types and optimised analysis time and sensitivity.

Evaporative light scattering detection (ELSD) is a good alternative to RID because the carbohydrate solutes, which do not possess chromophores that readily enable UV/Visible monitoring, are non-volatile - an essential attribute when considering ELSD. The system described here includes a binary gradient pump, autosampler, thermostated column compartment and an ELS detector. Software control and data reduction was made via a personal computer with appropriate software. While many HPLC systems are in use in a wide variety of applications worldwide, ELS detectors are unfamiliar to many users, with respect to the principle of operation and typical applications. Most HPLC detectors have an optical flow cell in which the solution-based measurements are made. The ELS detector, however, is quite unique by comparison. The three main steps used within ELSD technology are shown in Figure 1.

The objective of this study was to provide a straightforward analysis of

carbohydrates found in several fruits. The carbohydrates analysed were fructose, glucose, sucrose and maltose. In addition, sorbitol was also considered because this solute is present in some juices and can be analysed using the same system. Using the new Agilent 1200 Series ELSD the sensitivity for carbohydrates was found to be in the range of 1 to 10 nanograms on-column under comparable conditions (Figure 2). In addition, the design of the Agilent detector makes it ideal for analysis of coarse mixtures. From nebulisation to detection, parameters are optimised for a contamination-free process. Working with coarse mixtures or complex matrixes is now possible, without causing damage to the detector.

The gradient elution was found to perform the analysis in the shortest duration. To protect the carbohydrate separation column and to avoid any contamination, a short C8 pre-column was used. A water/acetonitrile gradient was used to separate the carbohydrates. The four selected components (fructose, sorbitol, glucose, sucrose) were separated in less than 12 minutes with complete resolution using a linear gradient pattern (Figure 3). For such an analysis, sensitivity is not an issue because the carbohydrates were present at high levels in juices. The injected masses on column were in the μg range.

Linearity tests were also performed using the Agilent 1200 Series ELSD. The standard flow nebuliser was chosen accordingly to the flow rate (1mL/min). Five nebulisers are available for the Agilent 1200 Series ELSD and each nebuliser is optimised to provide the best sensitivity and the best repeatability for a selected flow rate range. For quantification, the best choice was to plot the logarithm of area against the logarithm of injected mass. This provided a linear relationship with a good correlation coefficient, typically close to r2 > 0.99 (Figure 4). As a demonstration, this process was successfully applied to the separation of glucose, fructose, sorbitol and sucrose.

An injected mass range from 1μg to 30μg was chosen, which corresponded to the levels found in the 1/100 or 1/1000 dilutions. Four juices were purchased in a local supermarket (orange, grape, pineapple and apple). These were either pure juices, or juices diluted from concentrates. All juices were filtered using a 0.2μm filter and diluted to 1/100 or 1/1000, depending on the concentration level of carbohydrates. Quantification was performed successfully based on the quantitative data established using the carbohydrate standards. The results expressed in milligram per litre in each juice are given in Table 1.

ELSD offers an opportunity to easily monitor separations of compounds that do not readily absorb UV light, and as a result a variety of classes of materials that are candidates for ELSD detection can be considered. Notably, many low molecular weight polymers can be detected. Triglycerides, phospholipids, sterols and phytosterols can also be monitored.
An original separation method has been developed for the separation of carbohydrates in coarse fruit juice mixtures. It was demonstrated that the use of a rapid filtering process before dilution of the sample and the use of a short reverse phase C8 pre-column were sufficient to protect the column and avoid matrix effects. The Agilent 1200 Series ELSD was used to analyse the carbohydrate mixtures and generate quantitative data. The correlation coefficients of the quantitative plots were excellent, confirming the quantitative capability of evaporative light scattering detection. Combining an optimised separation with the Agilent 1200 Series ELSD provides for acquisition of quantitative data from coarse fruit juice mixtures, without the need for extensive sample pretreatments.