1.1.5. Analysis
Most methods for the analysis of 3-MCPD focus on the trace analysis at microgram per kilogram levels in various food matrices, which is relatively complicated (Wenzl et al., 2007). The three main physical characteristics that contribute to these complications have been attributed to the absence of a suitable chromophore, a high boiling-point and a low molecular weight (Hamlet et al., 2002a). Initial methods that were developed for the determination of chloropropanols without derivatization showed low sensitivity (Table 1.1). Because of the absence of a chromophore, approaches based on high-performance liquid chromatography with ultraviolet or fluorescence detection cannot be applied, and only one such method with refractive index detection that has been used to study the kinetics of 3-MCPD formation in model systems appears to be unsuitable to determine trace quantities of the compound in food matrices (Hamlet & Sadd, 2002).
Direct analysis by gas chromatography (GC) without derivatization is also restricted. The low volatility and high polarity of 3-MCPD give rise to unfavourable interactions with components of the GC system that result in poor peak shape and low sensitivity. For example, during GC, 3-MCPD can react with other components of the sample to form hydrochloric acid in the presence of water, and with active sites in the column and non-volatile residues in the column inlet (Kissa, 1992). Interferences may also arise from the reaction of 3-MCPD with ketones contained in the matrix to form ketals (Kissa, 1992). Peak broadening and ghost peaks were observed with GC-based methods for the analysis of underivatized 3-MCPD (Rodman & Ross, 1986).
The low molecular weight of 3-MCPD aggravates detection by mass spectrometry (MS) because diagnostic ions cannot be distinguished reliably from background chemical noise. Due to these apparent limitations, the methods based on direct GC (e.g. Wittmann, 1991; Spyres, 1993) are more or less obsolete, and, because of their high limits of detection, are unsuitable to control maximum levels of 3-MCPD (see Section 1.4).
Xing & Cao (2007) developed a simple and rapid method applied capillary electrophoresis with electrochemical detection. However, its sensitivity appears to be insufficient to determine contents in the lower microgram per kilogram range found in foods.
None of the methods that use underivatized analytes is of sufficient sensitivity or selectivity to determine low microgram per kilogram levels in foodstuffs, nor is derivatization using sylilation with bis(trimethylsilyl)trifluoroacetamide (Kissa, 1992; Bodén et al., 1997), the detection limits of which were above 0.02 mg/kg using MS.
In combination with GC-MS, the three most common derivatives that give adequate sensitivity and selectivity are: (1) cyclic derivatives from the reaction with n-butylboronic acid or phenylboronic acid (PBA) (Rodman & Ross, 1986; Pesselman & Feit, 1988); (2) heptafluorobutyrate derivatives from heptafluorobutyrylimidazole (HFBI) or heptafluorobutyric anhydride (van Bergen et al., 1992; Hamlet, 1998; Chung et al., 2002); and (3) cyclic ketal derivatives from ketones (Meierhans et al., 1998; Dayrit & Niñonuevo, 2004; Rétho & Blanchard, 2005). These methods are summarized in Table 1.1. For further details on the derivatization of 3-MCPD, see Wenzl et al., (2007).
Of the different procedures, the PBA derivatization method is the most common. For example, it is used as a German reference method for food (Anon., 1995). An advantage of PBA derivatization is that no sample clean-up is required because PBA reacts specifically with diols to form non-polar cyclic derivatives that are extractable into n-hexane. The disadvantage of PBA is that other chloropropanols, such as 1,3-dichloro-2-propanol (1,3-DCP) cannot be determined simultaneously using this method. Sensitivity can be further improved by the application of triple quadruple MS/MS (Kuballa & Ruge, 2003). Sample preparation may possibly be improved by headspace solid-phase microextraction (Huang et al., 2005).
HFBI/heptafluorobutyric anhydride derivatization is also very commonly applied, although it is less selective than that with boronic acids. The procedure has been validated by a collaborative trial (Brereton et al., 2001). Repeatability ranged from 0.005 to 0.013 mg/kg and reproducibility from 0.010 to 0.027 mg/kg. The validation of the method was judged to be satisfactory and the method was adopted by the Association of official Analytical Chemists International as an official method (Brereton et al., 2001). The method was also adopted as European norm EN 14573 (European Standard, 2005). The HFBI method was found to be more labour-intensive than the PBA method but has the advantage of analysing both 1,3-DCP and 3-MCPD simultaneously during the same GC-MS run. The procedure can also be used with little modification to analyse blood and urine samples of rats in the context of toxicological studies (Berger-Preiss et al., 2010).
Currently, only a few methods exist to analyse so-called ‘bound’ 3-MCPD, which is a 3-MCPD ester bound with fatty acids. Unhydrolysed MCPD esters can be analysed directly by extraction into an organic solvent, clean-up by a preparative thin-layer chromatography (Davídek et al., 1980) and analysis using GC-MS (Hamlet & Sadd, 2004). More commonly, bound 3-MCPD is released from the esters and analysed in free form, through enzyme hydrolysis with a commercial lipase from Aspergillus (Hamlet & Sadd, 2004), interesterification of the sample with sulfuric acid (Divinová et al., 2004), or cleavage with sodium methoxide (Weißhaar, 2008) or methanolic sodium hydroxide (Kuhlmann, 2010).