Effective SPE Strategies for LC-MS Determination of Sudan Dyes in Chili Products

This application note highlights the LC-MS analysis of sudan dyes in chili products.

Benefits

• The Sep-Pak Alumina B SPE Procedure provides enrichment and clean-up for oils and oily matrices such as chili sesame oil and dried chili products
• The Oasis MAX SPE procedure provides enrichment and clean-up for water-based samples such as chili sauces
  

Recently there have been numerous reports regarding the potentially dangerous use of Sudan dyes for colorizing food products (structures shown in Figure 1). For example, Sudan I is an intense orange dye useful as a coloring agent for plastics and other industrial products but not intended for use in foods. However, Sudan I has been found in red chili products and in tomato-based products sold for human consumption. Sudan dyes are aromatic azo compounds and considered to be genotoxic carcinogens. This has necessitated the development of improved methods for their identification in foods.

Two complementary SPE procedures have been developed for rapid isolation and clean-up of Sudan dyes in chili and chili products. The first, more appropriate for non-oily matrices such as fresh chilies, is a mixed-mode anion-exchange procedure using Waters Oasis MAX sorbent. The second, more appropriate for oily matrices and dried chilies, is a normal-phase clean-up using Waters Sep-Pak Alumina B. LC-MS analysis is accomplished with positive electrospray ionization. In most cases, the enrichment and cleanup obtained from SPE allows for mass spec screening (SIR) and confirmatory analysis with LOQ below 10 μg/kg using tandem LC-MS/MS (MRM). The types of chili products studied are shown in Figure 2. Some potential interferences removed using these SPE methods are shown in Figure 3.

The calibration curve prepared in spiked chili sauce was linear in the range from 8 to 100 μg/kg (R² = 0.995).

Oasis MAX (3cc, 60 mg)

Pre-extraction: Chili products (1 g) are homogenized and extracted with 10 mL acetone. A 1 mL aliquot is diluted to 5 mL with aqueous NaOH (to pH 11).

Condition: 2 mL ethyl acetate 2 mL methanol, 1 mL 0.1 M NaOH, 2 mL water

Load: 5 mL of diluted acetone pre-extract

Wash: 2 mL 70% methanol in water,1 mL 1 M NaOH in water, 2 mL methanol, 1 mL ethyl acetate

Elute: 2 mL 89:9:2 ethyl acetate/MeOH/formic acid - evaporate and reconstitute in 90:10 acetonitrile/water (200 μL)

Note: Polar phenolics such as capsaicin are not retained by ion-exchange at pH 11 and are removed with wash 1. Wash 2 (1 M NaOH) ionizes the retained Sudans prior to wash 3 and 4 (to remove non-polar neutrals and bases).

Results from spiked chili sauce (n = 6, 80 μg/kg)

Sudan I         83% recovery (9% RSD)
Sudan II        83% recovery (1% RSD)
Sudan III       77% recovery (3% RSD)
Sudan IV       75% recovery (4% RSD)

A representative LC-MS chromatogram is presented in Figure 4.

Pre-extraction: Chili oils (0.1 g) are diluted to 1 mL with hexane. Chili products (1 g) are homogenized and extracted with 10 mL acetone. A 1 mL aliquot is evaporated to complete dryness and the residue is taken up in 1 mL hexane.

Condition: 2 mL methanol, 2 mL ethyl acetate, 3 mL hexane

Load: 1 mL of pre-extract in hexane

Wash: 3 mL hexane, 1 mL ethyl acetate

Elute: 4 mL 90:10 ethyl acetate/methanol - evaporate andreconstitute in methanol (200 μL)

Note: Fats are completely removed with the hexane and ethyl acetate washes. Carotenoids are removed with the ethyl acetate wash. The Sudan dyes are eluted with 10% methanol in ethyl acetate while more polar interferences, such as capsaicin, are retained.

Results from spiked chili oil (n = 6, 80 μg/kg)

Sudan I          99% recovery (11% RSD)
Sudan II         91% recovery (11% RSD)
Sudan III        93% recovery (6% RSD)
Sudan IV        122% recovery (11% RSD)

A representative LC-MS chromatogram is presented in Figure 5.

Considerations for SPE

• Sudan dyes are well retained on all Oasis sorbents by reversed-phase
  
• Oasis MAX allows for ion-exchange retention at pH 13
  
• Sudan dyes are also well suited for normal-phase retention (i.e. on Alumina B)

Analysis of Chili Products Obtained from Local Stores

Seven powdered products and two oils were analyzed using Sep-Pak Alumina SPE for sample preparation. Two water-based chili sauces were analyzed using  Oasis MAX SPE for sample preparation. Results are summarized in Table 1 and representative chromatograms are shown in Figures 6 and 7.

The Sep-Pak Alumina B SPE Procedure provides enrichment and clean-up for oils and oily matrices such as chili sesame oil and dried chili products. The Oasis MAX SPE procedure provides enrichment and clean-up for water-based samples such as chili sauces.

In this study, five out of the 11 commercial samples analyzed were found to be contaminated with Sudan dyes. Three dried chili products contained greater than 250 μg/Kg (ppb) of Sudan I; one sample measured 88 mg/kg (ppm) of Sudan I. Two chili sauces contained over 80 μg/kg of Sudan II.

 The high incidence and concentration of Sudan dyes in the test samples provides evidence of the need for further investigation of Sudan dyes in U.S. food products.