## 5 Analytical Methods
### Other Techniques  
Paper spray mass spectrometry has been used for the analysis of 4(5)MEI in caramel and beverage samples [55]. The solution of the sample was applied to a cellulose chromatography paper followed by the application of a high voltage (+2.5-3.5 kV). The researchers evaluated three different types of mass spectrometers for different purposes (linear ion trap, triple quadrupole, and orbitrap). The triple quadrupole mass spectrometer provided a detection limit of 5 pg \(\mu\)L\({}^{-1}\), while LOQ was 20 pg \(\mu\)L\({}^{-1}\). The analytical results from the linear ion trap MS were similar to those obtained from the triple quadrupole, while the orbitrap mass spectrometer provided a detection limit of 100 ng mL\({}^{-1}\). To determine the 4(5)MEI in caramel color, [PERSON] et al. [54] developed a capillary electrophoresis method. An uncoated fused-silica capillary of 75 \(\mu\)m i.d. was utilized with a length of 40 cm. The running electrolyte consisted of 160 mmol L\({}^{-1}\) phosphate plus 30% acetonitrile at pH 2.5, which was adjusted with triethylamine. The method provided very good recoveries (89.1 \(\pm\) 2.0-106.9 \(\pm\) 1.8%), and a LOQ at 0.54 mg L\({}^{-1}\). [PERSON] et al. [57] used high-performance cation exchange chromatography with pulsed integrated amperometric electrochemical detection (HPCEC-PAD) for the analysis of 4(5)MEI in caramel color and beverages. Samples were SPE pretreated and filtered through a clarification kit of 0.45-\(\mu\)m pore size. Separation was achieved on a CS12A cation exchange column (250 \(\times\) 4 mm) and a CS12A guard column (50 \(\times\) 4 mm) utilizing the isocratic elution of 10 mmol/L methane sulfonic acid for 50 min. The recoveries obtained were 93.7-98.3%, while the LOQ was lower compared to the capillary electrophoresis method (0.045 mg L\({}^{-1}\)). An amino trap column coupled with pulsed amperometric detection (AMTC-PAD) has been employed by [PERSON] et al. [89] for the determination of 4(5)MEI in beverages. Samples were pretreated with SPE using SCX Disc 15 mg/3 mL cartridges and filtered through a 0.45-\(\upmu\)m pore size clarification kit. An amino trap column (4 \(\times\) 50 mm) was used with an isocratic elution of 100 mmol L\({}^{-1}\) NaOH for 60 min. The LOQ obtained was comparable with the previously described HPCEC-PAD method (0.045 mg L\({}^{-1}\)) and the recovery ranged from 95.1-103.4%. An indirect competitive enzyme-linked immunoassay(ic-ELISA) was developed by [PERSON] et al. [90] based on the monoclonal antibody for the detection of 4(5)MEI in caramels. Ovalbumin or bovine serum albumin and the 4(5)MEI hapten were used for the preparation of artificial antigens. The cross-reactivity of 4(5)MEI structural analogues was less than 5.62%. The linear detection range was 0.64-20.48 mg L\({}^{-1}\) and the recoveries were between 88.69% and 114.09%. [PERSON] et al. [56] developed a novel method employing cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-sweeping-MEKC). The samples analyzed were soft drinks and alcoholic beverages, which were diluted with 0.05 mM H\({}_{3}\)PO\({}_{4}\) and injected for direct analysis in the online CSEI-sweeping-MEKC. The researchers also prepared an SPME adsorbent by incorporation of activated carbon into a monolithic column with poly(butyl methacrylate-co-ethylene dimethacrylate). This adsorbent was used to extract 4(5)MEI from the samples with subsequent analysis by CSEI-sweeping-MEKC. The LOQs were 20 and 125.9 \(\upmu\)g L\({}^{-1}\) for the CSEI-MEKC method and the SPME CSEI-MEKC method, respectively, and the recoveries ranged from 94.0 to 94.8%. A fluorescence-based immunomodographic assay has been developed by [PERSON] et al. [58] for the quantitative detection of 4(5)MEI in caramel color. The authors used as a probe conjugates of fluorescent microspheres and the 4(5)MEI monoclonal antibody. The method recovery ranged from 82.85 to 102.31% and the LOQ was 0.6 mg L\({}^{-1}\). A THz technique was utilized by [PERSON] et al.