3.1. Optimization of UPLC conditions
The peak time of 15 dyes was controlled from 2 to 16 mins to balance the chromatographic column. A mixture of acetonitrile and methanol was used as mobile phase A to increase elution capacity. The solution of ammonium acetate and formic acid was used as mobile phase B uses to ionize both positive and negative ions. The result of the UPLC conditions was shown in Table 1.
3.2. Optimization of MS conditions
In this experiment, the standard solution of a single dye was diluted to 500 ug/L, and the data were collected in the mass spectrometer. The result of the MS conditions was shown in Table 2. First, the positive ion mode was adjusted to find the parent ion of the standard in this scanning mode. Then, the corresponding daughter ions were found by gradually increasing the collision voltage. After that, the 15 dyes were divided into five different channels to enter the mass spectrometer to improve the sensitivity of the instrument.
3.3. Optimization of the polyamide membrane purification method
3.3.1. SEM of the membrane
Fig.1 (a) showed the surface electron micrograph of the polyamide membrane, and it can be seen that the surface is smooth. The picture of cross-section showed that many "finger" pores were existing in the membrane, which is beneficial to the purification of the sample, as shown in Fig.1 (b). PSA as the adsorbent existing in the surface and pores of membrane will adsorb the illegally industrial dyes. So the process of pretreatment with the membrane need two procedure, adsorption the illegally industrial dyes to the membrane and then elution them from the membrane. The schematic of purification by the polyamide membrane was shown in Fig.2.
3.3.2. Optimization of polyamide addition in the membrane
A series of membranes were made to investigate the influence of PSA content to the purification and extraction effect, the PSA content of them were 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt% and 80 wt% respectively.
The results were shown in Fig.3. It can be seen that the recovery of the illegally industrial dyes enhanced with the increase of PSA content. It may be caused by the more complete adsorption of the membrane to illegally industrial dyes. When the content of PSA content increased to 80%, the recovery can meet the determination requirements. For the consideration of much PSA may bring some defect to the performance of membrane, so the optimal PSA content was set as 80%.
3.3.3. Optimization of elution solvent
Different elution solvent in the pretreatment of millet were tested to seek the suitable condition for the fully elution. First 5 mL extract solution was taken to be absorbed by the polyamide membrane, then eluted with different elution solvents to investigate the elution effect. Methanol, acetonitrile, ammoniated methanol, acetone, n-hexane, isopropanol, dichloromethane:methanol = 9:1 and isopropanol:dichloromethane = 9:1 were tested.
It can be found that three of them can be used as effect elution solvents with the recovery of the method between 70%-120% to meet the detecting requirements. Acid violet 9, acid yellow 36 and acid orange II were eluted by ammoniated acetonitrile (5 ml) successively. Sudan Red G and Sudan Red I were eluted by N-hexane of 5 ml successively. The other 10 industrial dyes were eluted with 5 ml of acetonitrile.
3.4. Linear equation and linear range of the method
Under the optimal pretreatment and instrument conditions, a mixed standard solution of 15 kinds of dyes was added to the blank millet sample. Linear equation and linear range of the method were investigated. The results are shown in Table 3. R2 of standard curve were all greater than 0.99.
3.5. Liquid chromatography-tandem mass spectrometry
Fig.4 showed UPLC-MS/MS chromatograms of the 15 industrial dyes. The ionization of 15 industrial dyes in the positive mode electrospray ion source was examined. The method of UPLC-MS/MS was highly selective for monitoring specific MRM and was effective in reducing the risk of false positives. The 15 industrial dyes were separated completely by UPLC-MS/MS.
3.6. Precision and detection limit of the method
The mixed standard solution of 15 dyes were add to millet and sausage samples and then be pretreated by the membrane as optimized previously. Six needles of each purification were repeated in the UPLC-MS/MS to obtain the instrument's precision (RSD). The detection limit of each industrial dyes was calculated according to 3 times S/N, and the quantitative limit was 5 times S/N. The results were shown in Table 4. The LOD and LOQ of these industrial dyes were ranged from 0.0003 to 0.70 and from 0.0017 to 3.35 respectively.
3.7. Results of recovery and precision of polyamide membrane extraction
Sausage, bacon, millet and corn flour were chosen as meat and grain matrix to investigate the recovery of the 15 industrial dyes. These samples were pretreated by polyamide membranes and analyzed by UPLC-MS/MS. The results were shown in Table 5. The recovery of the dyes was between 71% and 126%, and the precision was between 3 and 13. The method of polyamide membrane as pretreatment combined with UPLC–MS/MS was confirmed reliable and effective in the determination of industrial dyes for grain and meat products.
3.8. Detection of actual samples
Three kinds of grain products (purple rice, soba noodles, mung bean) and three kinds of meat products (sausage, pig head meat, ham sausage) were randomly selected from the market to detect the 15 illegal industrial dyes. The samples were pretreated by polyamide membrane made by ourselves and then analyzed with UPLC–MS/MS. No illegal industrial dyes were found in the actual samples.