Sher Mohammed, Nidhal Meena
Extraction and HPLC analysis of potato sprout suppressant chemicals.
PhD thesis, University of Glasgow.
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In the UK, up to six million tonnes of potatoes are produced annually and more than half of this production is stored for the fresh market and food processing. To maintain potato quality from sprouting, chlorpropham (CIPC) is currently used as the main sprout suppressant in commercial potato stores. Questions have been raised about the safety of application of this compound in potato stores due to increasing concern about the toxicity of its residue and degradation products mainly 3-chloroaniline (3-CA) on the potato tubers. To date, there is no realistic replacement of CIPC for inhibiting sprouting of potatoes destined for processing. Searching for alternatives is crucial particularly as most supermarkets demand foods free of chemicals. The sprout inhibitor 1,4-dimethylnaphthalene (1,4-DMN) can be a suitable replacement for CIPC as it is naturally occurring in the potato and currently used in many countries in the world. To introduce this compound to the UK for commercial use, many investigations must be conducted to ensure its safety for human health and the environment. This study intended to focus on the determination of the residue level of 1,4-DMN, CIPC and its metabolite 3-CA in potato and water samples, hence developing analytical methods was required as a preliminary step.
In this study, three HPLC systems were used for validating a separation method for the analysis of 1,4-dimethylnaphthalene and its internal standard 2-methylnaphthalene (2-MeN). Under the same chromatographic conditions, all these systems achieved excellent separation on a Jones-ODS column (Hypersil ODS 5 µm, 250 mm x 4.6 mm) at ambient temperature isocratically using 70% acetonitrile as mobile phase at a flow rate of 1.5 mL/min, 20 µL injection volume, a run time of 10 minutes and a detection wavelength of 228 nm. All three systems showed high precision (RSD% < 1%), good linearity of the calibration curves at two concentration ranges (0.02 – 0.1 and 0.2 – 1.0 µg/mL) of each 1,4-DMN and 2-MeN with coefficient of determination (R2) of the regression line of 0.990 or more. The best system SpectraSERIES UV100-autosampler system was selected for the remainder of this research as it offered lower values for both the limit of detection (LOD) (0.001 – 0.004 µg/mL) and the limit of quantification (LOQ) (0.002 – 0.013 µg/mL) for both compounds.
A second isocratic reversed phase HPLC-UV method was developed and validated for analysis of 1,4-DMN and 2-MeN using methanol as a substitute solvent for standards and mobile phase preparations to overcome the problem of a global shortage of acetonitrile during 2008 – 2009. The best separation was achieved on the Phenomenex® (ODS-2 250 mm x 4.60 mm 5 µm Sphereclone) column using 90% methanol as mobile phase at a flow rate of 1.5 mL/min and a 6 minute run time. The method was validated producing good precision, linearity and low values of LOQ (~ 0.001 µg/mL).
A straightforward and rapid isocratic HPLC-UV method was developed and validated for the simultaneous analysis of both CIPC and its degradation product 3-CA using methanol as a solvent and propham (IPC) as an internal standard. To achieve high resolution of the three compounds, the chromatographic conditions selected were: Phenomenex® column (ODS-2 250 mm x 4.60 mm 5 µm Sphereclone), 62% methanol at a flow rate of 1.5 mL/min, detection wavelength of 210 nm and a 15 minute run time. Method validation confirmed good precision, acceptable linearity and low values of LOD (~ 0.01 µg/mL) and LOQ (~ 0.04 µg/mL) for CIPC and 3-CA. These proposed HPLC methods are suitable to apply for the determination of the studied compounds in both potatoes and water samples.
Quantitative laboratory analysis of 1,4-DMN, 2-MeN , CIPC and 3-CA in water solutions showed acceptable standard preparations in water with good precision and linearity and lower values of LOD and LOQ close to those obtained in organic solvent preparations. An adsorption study of 1,4-DMN and 2-MeN on laboratory ware showed that glass materials were acceptable to use whereas there was a considerable adsorption to plastic containers and filters. In contrast, 3-CA showed no adsorption onto any of the laboratory ware tested. CIPC also showed good recoveries with most of the materials tested.
In reviewing the literature, no suitable published method for the simultaneous determination of CIPC and its metabolite 3-CA in potato peel was found. A simple analytical method was developed based on methanol-soaking overnight extraction coupled with HPLC-UV for analysis of CIPC and 3-CA in potato samples using IPC as internal standard. The method was validated and the calculated limit of quantification was 0.01, 0.05 and 0.02 µg/g in whole tuber for CIPC, IPC and 3-CA respectively. The efficiency of the method reported recovery values of up to 90% for both CIPC and IPC through spiking organic potato peel at three spiking levels of 0.8, 8.0 and 80 µg/g. By contrast, 3-CA recoveries offered very low values of 10 and 23% at concentration levels of 8.0 and 80 µg/g respectively and no peak was detected at the lower level of 0.8 µg/g. This method was compared with the routine Soxhlet-GC method used for the analysis of the residues of CIPC in potato samples at the University of Glasgow laboratory and gave results approximately 23% higher residues of CIPC. This new method at this stage was suitable to extract CIPC in 20 potato samples daily. Nevertheless, an interesting finding was that despite the low recovery of 3-CA it was identified in treated potato samples. This unanticipated low recovery is noteworthy and indicates that the actual residue may be much higher.
A comprehensive study was made to improve the extractability of 3-CA from potato samples investigating parameters including potato variety, extracting solvent, extraction method, spiking procedure and different treatments for potato samples. All these experimental trials showed no recovery improvements, thus four possible mechanisms were suggested for poor recovery of 3-CA including volatilisation, reaction with potato components, enzymatic activity and ion exchange related to pH.
Under the laboratory work experimental conditions, no measurable loss of 3-CA by volatilisation was found. No reaction of 3-CA was found to occur with other potato components under the experimental conditions used. However, the Schiff base reaction and/or hydrogen bonding may link the amino group of 3-CA and some functional groups abundant in potatoes (e.g. carbonyl, quinone). This study also showed a potential role for enzymatic activity in the poor recovery of 3-CA. Using antioxidants or acidity to inhibit this enzymatic activity was shown to enhance the extractability of 3-CA. Binding of 3-CA to potato peel substrates by ion exchange is unlikely as the pKa value of 3-CA is lower than the pH of the potato. However, using sulphuric acid combined with methanol as an extracting solution improved the recovery. Optimising the extraction process showed that using a mixture of 1 M H2SO4 in 50% methanol as an extracting solution for 24 hours at 50 ºC improved the extraction recovery of 3-CA up to 85%. This final extraction method was applied for the determination of the residues of both CIPC and 3-CA in commercial potato samples which had received many applications of CIPC, thus reporting high residue values. Additionally, potato samples were taken from different UK stores for the storage season 2010 – 2011 which had received CIPC application at high and low temperature (450 ºC and 270 ºC respectively) fogging. Analysis of these potato samples showed no significant difference between high and low temperature for the first application of CIPC for both residues of 3-CA and CIPC. A significant increase in both compounds was found between the first and second application at 270 ºC indicating a possible build up with time during storage.
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