Food additives, HPLC analysis

Scotter, M.J., Castle, C., Roberts, D., Method development and HPLC analysis of retail foods and beverages for copper chlorophyll (E141[i]) and chlorophyllin (E14[ii]) food colouring materials. Food Additives and Contaminants, 22,1163, 2005. 

For reference methods, HPLC with various detectors has become the standard reference technique for analysis of food additives, but new developments in this area are mainly linked to detector technology. Diode array detectors have not totally met the expectations of food analysts in terms of their specificity and LC-MS is likely to fill the gap. Specific detection with biosensor chips may also have a future for certain analyses. The use of combined LC-MS/DAD systems is... 

Recently we published data that even in countries with excellent food sources and availability, insufficient vitamin A supply will occur (Schulz et ah, 2007). The aim of this trial was to analyze vitamin A and p-carotene status and investigate the contribution of nutrition to vitamin A and p-carotene supply in mother-infant pairs of multiparous births or births within short birth rates. Twenty-nine volimteers aged between 21 and 36 years were evaluated for 48 hours after delivery. In order to establish overall supply, retinol and p-carotene were determined in maternal plasma, cord blood, and colostrum via HPLC analysis. A food frequency protocol was obtained from all participants. Regardless of the high-to-moderate socioeconomic background, 27.6% of participants showed plasma retinol levels below 1.4 pmol/liter, which can be taken as borderline deficiency. In addition, 46.4% showed retinol intake <66% of RDA and 50.0% did not consume liver at all, although liver contributes as a main source for preformed retinol. Despite a high total carotenoid intake of 6.9 3.9mg/day, 20.7% of mothers showed plasma levels <0.5 pmol/liter p-carotene. 

The application of HPLC in routine environments, like pharmaceutical, food, or environmental analysis and particularly quality assurance, makes not only great demands on the robnstness of HPLC hardware, comprising pumps, column thermostats, and detection units, bnt in addition to the column reproducibility. Column reproducibility can be investigated at different levels of complexity Run-to-run reproducibility compares consecutive chromatographic runs, whereas long-term stability describes the column variance over several hundreds of injections. Column-to-column (batch-to-batch) reproducibility finally explores the match of independently fabricated chromatographic columns. Column characteristics that are routinely consulted for the determination of the robustness are retention, selectivity, column efficiency, and peak symmetry. 

K Saag. Determination of food additives by HPLC. In R Macrae, ed. HPLC in Food Analysis. New York Academic Press, 1988, pp 197-210. 

One of the major advantages of HPLC in food additives analysis is the comparably simple possibility of automation. When there are many samples to analyze and quantify in routine analysis, an autoinjector usually is the additional component of choice. An autosampler may help to reduce costs, and the instrument may be left to run analyses overnight. Some autoinjectors can also be used to derivatize samples by adding the relevant reagent before injection into the chromatographic system (5). 

One limiting component of HPLC systems for the analysis of different food additives is the choice of the detector. In recent years, monitoring peak elution via the absorption of ultraviolet (UV) light has been the most common method, because the vast majority of compounds have some absorbance in the UV or the visible region. The popularity of this detection mode is primarily due to its sensitivity toward a large number of constituents in the range of 210-280 nm. 

Veerabhadrarao et al. (76) used reverse-phase HPLC for the determination of some food additives (acesulfame, saccharine, BA, p-hydroxybenzoic acid). The samples (beverages, tomato sauce) were diluted and then separated on a /rBondapak CJg column with methanol/acetic acid/water (20 5 75) or (35 5 60) as mobile phases. The determination was done at 254 nm. Recoveries varied from 98 to 106% for direct analysis and from 91.6 to 101.8% for extraction of samples (76). 

The fabrication of imprinted monolithic solid-phase microextraction fibres has been developed for the selective extraction and preconcentration of diacetylmorphine and its structural analogues, triazines, bisphenol A, anaesthetics, and antibiotics followed by GC or HPLC analysis [156,163,179,196,197]. In addition, the on-line coupling of the imprinted monolith as a preconcentration column with a conventional analytical column has been proposed for the enrichment and cleanup of environmental and food samples [163]. However, at present, the capacity of the imprinted fibres and thus the degree of recovery of analytes are very variable and obviously need some improvement. For example, the recoveries of triazines after SPME with an imprinted monolith prepared by in situ polymerisation of MAA as... 

Puech, L., Dragacci, S., Gleizes, E., and Fremy J-M. (1999) Use of immunoaffinity columns for clean-up of diarrhetic toxins (okadaic acid and dinophysistoxins) extracts from shellfish prior to their analysis by HPLC/fluorimetry. Food Additives and Contaminants, 16, 6, 239-251. 

Procedures based on separation techniques such as HPLC and IC have been developed for single element analysis for the following two reasons. The first reason is to remove interferents in complicated sample matrices that can give rise to incorrect results, in particular for trace analysis in samples with a high organic content, such as the determination of total iodine in egg products. The second reason is to differentiate the total and free forms of a specific element, such as the determination of the free iodide ion and bounded iodine in food additives. The free iodide ion is determined by direct sample injection into the IC column, whereas the total iodine content is determined after oxygen flask combustion. Thus, both the free and bounded forms of iodine in food samples can be determined. 

HPLC has been used increasingly in the analysis of food samples to separate and detect additives and contaminants. HPLC can separate a large number of compoimds both rapidly and at high sensitivity, reduce separation times, and reduce the volume of sample needed. HPLC is ideally suited for compounds of limited thermal stability, but requires sample pretreatment such as extraction and filtration. In addition, HPLC requires careful selection of mobile phase and sample pumping rate [24]. 

PL was determined under reaction conditions 1. A food sample (5 pL) was added to reaction mixture 1 to start the PLDH reaction. After 1 h, the reaction was stopped with HCl. Then, the mixture was filtered through a Dismic 13 syringe filter (pore size, 0.2 pm Advantech, Tokyo, Japan). The filtrate (100 pL) was applied to the HPLC column. The amount of sample could be increased up to 200 pL depending on the PL content in the sample the amount of water should be decreased to make the volume of the reaction mixture 380 pL. HCl was added before addition of the sample to obtain a control (no reaction) reaction mixture. For determination of the yield, a food sample containing a definite amount of standard PL was also analysed. The amount was calculated based on the results of preliminary analysis of the PL content of the food sample. For analysis of urine, 8 pmol of standard PL was added to reaction mixture 1. The yield was calculated as follows ... 

Llewellyn LE, Doyle J, Jellett J, Barrett R, Alison C, Bentz C, Quilliam M (2001) Measurement of paralytic shellfish toxins in molluscan extracts comparison of the microtitre plate saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay. Food Addit Contam 18 970-980... 

Kritsunankul et al. [76] proposed flow injection online dialysis for sample pretreatment prior to the simultaneous determination of some food additives by HPLC and UV detection (FID-HPLC). For this, a liquid sample or mixed standard solution (900 pL) was injected into a donor stream (5%, w/v, sucrose) of a FID system and was pushed further through a dialysis cell, while an acceptor solution (0.025 mol/L phosphate buffer, pH 3.75) was held on the opposite side of the dialysis membrane. The dialysate was then flowed to an injection loop of the HPLC valve, where it was further injected into the HPLC system and analyzed under isocratic reversed-phase HPLC conditions and UV detection (230 nm) (Figure 24.6). The order of elution of five food additives was acesulfame-K, saccharin, caffeine, benzoic acid, and sorbic acid, with an analysis time of 14 min. This system has advantages of high degrees of automation for sample pretreatment, that is, online sample separation and dilution and low consumption of chemicals and materials. 

The synthetic colorants Quinoline Yellow and Sunset Yellow are determined individually or simultaneously with other food additives by several methods, mainly spectrophotometry and chromatography. At present, the most used technique for synthetic colorants determination is liquid chromatography. The most popular reported HPLC modes applied for synthetic colorants analysis are reversed-phase chromatography (RPC), ion pair chromatography (IPC), and ion chromatography (IC). In recent years, electrochemical techniques have attracted wide attention due to their characteristics such as low operational cost, rapid and sensitive detection, selectivity, and reproducibility. 

A method for the analysis of such dyes has been developed. The method is based on coupling of ionic liquid-based extraction with HPLC. In this way, Sudan dyes and Para Red in chib powder, chib oil, and food additive samples can be found. 

Liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) was introduced in the 1980s [1]. Today it has become a standard method for separation and characterization of nonvolatile compounds. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to ESI-MS is the method of choice for peptide and protein analysis, but also used for the characterization of contaminants, therapeutic drugs, and food additives [2-5], More than 75% of HPLC analyses are run on RP stationary phases, and a wide range of columns are available with various substituents of the silica matrix, base deactivation, endcapping, and column dimensions. 

Bohman and colleagues described a reverse-phase HPLC method for the quantitative analysis of vitamin A in food using the method of standard additions. In a typical example, a 10.067-g sample of cereal is placed in a 250-mL Erlenmeyer flask along with 1 g of sodium ascorbate,... 

Carmine extracted from cochineal insects is one of the most used natural colorings for beverages and other foods. Some representative articles refer to isolation and spectrometric analysis or the use of HPLC or capillary electrophoresis (CE) to separate and characterize all cochineal pigments. Its active ingredient, carminic acid, was quantified by rapid HPLC-DAD or fluorescence spectrometry. Carminic acid, used as an additive in milk beverages, was separated within 9 min using a high-efficiency CE separation at pH 10.0 after a previous polyamide column solid phase extraction (SPE), ... 

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