Analysis of Polyphenols in Apple Pomace using Gas Chromatography-Mass Spectrometry with Derivatization

Abstract

N, O-double (three methyl silane) trifluoro ethyl amide was used to derivatize polyphenols in apple pomace, and the resultant products were tested. The optimal derivatization conditions were then used in conjunction with the rapid analysis of polyphenols by gas chromatography-mass spectrometry. In the results, the following conditions for derivatization with N, O-double (three methyl silane) trifluoro ethyl amide were obtained : Polyphenols should be derivatized for 12 h at 37.5^oC with a mass ratio of derivatization reagent to sample being 10 to 1. After analysis through gas chromatography-mass spectrometry, 24 compounds were identified, 11 of which were phenolic compounds and organic acids. Furthermore, four isomeric compounds of epicatechin were separated out. The method proved to be easy to use and sensitive, and could be used to analyze unknown phenolic compounds.

• Apple pomace
• Phenolic compounds
• Derivatization
• Gas chromatography-mass spectrometry
• BSTFA

INTRODUCTION

Apple is one of the most widely consumed fruits and is propagated in global temperate regions,[¹] with China being the world’s largest apple producer. Apple pomace is the main by product during the processing of concentrated apple juice, accounting for 25–35% of the weight of dried apple.[^2,3] It is a rich in source of carbohydrate, pectin, crude fiber, and minerals,[⁴] simultaneously, and it can be treated as an excellent example of trash recycling.[⁵]

In recent years, research has focused on the by-products of concentrated juice processing, in particular the processing and utilization of apple pomace,[^6,7] where polyphenols are the main focus of attention.[⁸⁻¹²] Many pharmacological studies show that polyphenols have antioxidant, anticancer, anti-atherosclerosis, anti-hyperlipidemia, bacteriostasis, anticarious, and radiation protective properties.[^6,13−19] Phenolic compounds may also help prevent coronary heart disease and strokes as well as having other biological activities. Their antioxidant capacity is 50 times more than vitamin E and 20 times more than vitamin C.[^20,21] Therefore, it is important to undertake analysis of the polyphenols found in apple pomace.

Most of the analysis of polyphenols have heavily relied on high-performance liquid chromatography (HPLC), mass spectrometry (MS), and capillary electrophoresis.[²²⁻²⁶] However, the HPLC-MS method is time-consuming which leads to a loss of analytes. The gas chromatography-mass spectrometry (GC-MS) method is simple, fast, and has high detection sensitivity, nevertheless, because of the strong polarity of polyphenols, sample derivatization processing needs to be done first, and the whole process requires complicated sample preparation. During the process, residual moisture in the extract is prone to peak tailing.[²⁷] In this study, with N, O-double (three methyl silane) trifluoro ethyl amide (BSTFA) as a derivatization reagent, combining GC-MC with traditional processing, a rapid method of determining polyphenols in apple pomace through parameter optimization and testing of analytical conditions was established.

MATERIALS AND METHODS

Instruments and Reagents

Instrument: Shimadzu 2010 GC-MS, Rtx-5 capillary column (30m × 0.32mm × 0.5μm). Reagents: derivatization reagent: BSTFA, containing 0.1% of trimethylchlorosilane) was purchased from the Regis Corporation; Ethanol (analytical grade) and macroporous resin NKA-9 were provided by Reagent Co., Ltd (Beijing, China). The apple pomace (Guoguang apple) was obtained from Yuantong Fruit Juice Factory in Yan-tai, Shang dong province, China, and vacuumized before analysis.

Polyphenols Extraction

Ethanol (extraction solvent, 60%) was mixed with apple pomace in a 1:6 solid:liquid ratio of pomace:ethanol(g ml⁻¹). A 250 W ultrasonic processor was used for extraction at 60^oC. Extraction time –30min; filtration-rotary evaporation to obtain crude polyphenol extract; purification by NKA-9 macroporous resin at a flow rate of 1.0 ml min⁻¹; elution conditions: 70% ethanol concentration; elution flow rate of 1.0 ml min⁻¹; elution volume – 10 BV; rotary evaporation; sample freeze-dried to obtain the polyphenol powder.[²⁸]

Polyphenols Derivatization

The method used to obtain the polyphenol derivatives: 0.1 g of the powder was put into a sample bottle together with the serum, to make up to a volume of 2–3 ml, after which the sample bottle was closed with a rubber cap. Ten to twenty times the volume BSTFA was added and the sample was shaken and left to stand for 20 min.

The GC-MS Analysis Conditions

Shimadzu 2010 GC-MS, Rtx-5 capillary column (30m × 0.32mm × 0.5μm). Split ratio 20:1; column temperature program settings: Column initial temperature was 100^oC, heating rate was 10^oC min⁻¹, up to 300^oC, maintained for 5 min, making a total analysis time of 25 min; high purity helium was used as a carrier gas flowing at a constant 1 ml min⁻¹; interface temperature was 250^oC, inlet temperature was 280^oC, EI ion source temperature was 200^oC; ionization voltagewas 70 eV with a multiplier voltage of 350 V; scan range was 33–500 m/z; scan speed was 0.5 s/time.

RESULTS AND DISCUSSION

Derivatization Reagent of Choice

In a previous study, polyphenols with high polarities that did not undergo the derivatization process before direct analysis with GC-MS produced fewer peaks and a lower number of peaks than were characteristic of polyphenols.[²⁰] Therefore, if the aim is to analyze polyphenols in apple pomace with GC-MS, then to obtain good peak effects, it is necessary to first use a derivatization treatment to reduce the sample polarity.

Pre-treatment of samples has the following advantages: (a) it increases the number of target samples and samples of volatile impurities and thus helps meet the requirements of GC; (b) it reduces the sample and the adsorption activity of the impurities in the sample (e.g., by removing active hydrogen atoms from the sample, thus changing the coordination properties between the sample and the stationary phase); (c) it allows more qualitative information to be obtained; (d) it improves the separation of compounds such as isomers and opticalisomers, which have similar properties.[²⁹⁻³¹]

Commonly used derivative methods include the silane derivative method and the esterification and acylation method. When the samples contain hydroxyl (-OH), amino (-NH2), and thiol (-SH) groups, the silane method is usually used. To reduce errors caused by derivatives, derivatization reaction steps should be minimized in order to improve the speed.[³²⁻³⁵] Therefore, this study increased the testing time of silane as it is considered the strongest of the BSTFA derivatization reagents.

Optimizing BSTFA Derivatization

Choice of derivatization reagent

Because the sample matrix consumes some of the derivative agents added, an excess of the chosen derivative agent needs to be added in order to complete the reaction.[³⁶] In this experiment, a sample and derivatization reagent mass ratios of 1:10 and 1:20 were chosen for gas chromatographic analysis. Figure 1 shows that the number of peaks and the peak height in Fig. 1a was significantly greater than seen in Fig. 1b. The sample and derivatization reagent mass ratio of 1:10, produced superior phenolic derivatives, which indicated that an excess of the derivatization reagent had a certain impact on the peak separation of the sample.

Figure 1 Effect of BSTFA sample volume on the gas chromatogram of the phenolic compounds found in apple pomace. (a) Mass ratio of sample to BSTFA is 1:10; (b) mass ratio of sample to BSTFA is 1:20; (c) control.

Derivatization Reaction Temperature

The effect of temperature on the derivatization reaction was crucial. If the temperature was too low, the derivatization reaction could not proceed to completion. If the temperature was too high, a small quantity of the sample components would decompose.[³⁷⁻³⁹] Due to the highly volatile nature of BSTFA, the derivatization reaction temperature was kept relatively low. Therefore, the two temperatures chosen for the derivatization reaction and the gas chromatography analysis were 10 and 37.5°C. The results are shown in Fig. 2. When comparing Figs. 2a and 2b, it can be seen that temperature does not have an obvious effect on the BSTFA derivatization reaction. Samples subjected to 37.5°C showed slightly higher maximum peaks than those samples subjected to 10°C. This indicated that at 37.5°C, the BSTFA derivatization reaction was more complete.

Figure 2 Effect of different reaction temperatures on the gas chromatogram of the phenolic compounds found in apple pomace. (a) Room temperature (20°C); (b) 37.5°C; (c) control.

Derivatization Reaction Time

Derivatives of the response must be completed, the reaction yield was not necessarily to 100%, but gave better reproducibility of measurements. The derivatives resulting from the reaction should be stable and not break down over time.[⁴⁰] Therefore, derivatization reaction times of 30 min and 12 h were chosen before the sample underwent GC analysis. The results are shown in Fig. 3, The spectra of the samples analyzed after 12 h were not significantly different from the samples analyzed after 30 min, but the result was slightly better than the 30 min response.

Figure 3 Effect of reaction time on the gas chromatogram of the phenolic compounds found in apple pomace. (a) 30 min; (b) 12 h; (c) control.

Qualitative Analysis and Composition of Polyphenols in Apple Pomace

The apple pomace samples contained a large number of polyphenols with chiral carbon atoms, resulting in a large number of isomers. The physical and chemical properties, together with the chromatographic behavior of these isomers, were very similar to the apple isolate, which made it very difficult to identify the polyphenols present.[⁴¹] The aim of the derivative pre-treatment was to improve the separation effect between these very similarisomers and optical isomer compounds. In this experiment, after derivatization by BSTFA, the polyphenols were qualitatively analyzed by GC-MS, Fig. 4 shows the derivatives observed from the total ion chromatogram. According to Fig. 4, 24 sharp peak shape and intensity high-resolution spectra were observed with a retention time range of 5–25 min. Using MS, the results showed 24 substances including 11 phenols and organic acid species. The peaks numbered 19, 21, 23, and 24 were left as unidentified epicatechins by MS. However, after derivatization pre-treatment, the four epicatechin stereoisomers were separated out. The remaining 13 substances were of the siloxane class, which suggested that they may be derived from the excess reagent added. Table 1 lists the phenolic compounds and organic acids found by the GC-MS analysis with a matching degree of 85–98%.

Figure 4 Total ion chromatogram of the phenolic compounds found in apple pomace after derivatization.

CONCLUSIONS

In this article, BSTFA derivatization with GC-MS was used to qualitatively analyze apple pomace polyphenols. It was found that the optimal pre-treatment conditions using BSTFA as derivatization reagent was a mass ratio of phenols to a derivative reagent of 1:10, a reaction derivative temperature of 37.5^oC, and a reaction derivative time of 12 h. After using the above derivatization pre-treatment method, followed by GC-MS analysis, 24 sharp peak shape and intensity high-resolution spectra were observed, which included 11 phenolic compounds and organic acid species. Furthermore, four epicatechin isomers were well separated. These results suggest that this BSTFA derivatization method, followed by GC-MS to identify the polyphenols present in the apple pomace, was a simple and straightforward method with a low sample consumption and would therefore be less expensive. This method is ideal to test for polyphenolic compounds of unknown composition.

Table 1 Phenolic compounds and organic acids found in apple pomace

Peak	Retention time/min	Polyphenolic compounds	Formula	Peak area /%
1	5.196	1,3-phenyl-1-acetone	C15H14O	0.61
4	11.133	N-(phenyl) methylene-β, 3,4-trihydroxy-	C15H15NO3	5.68
6	13.622	phenylethylamine	C3H3N3O3	3.48
8	14.405	2,4,6-trihydroxy-1, 3,5-triazine	C6H14O6	0.40
9	14.815	d-mannitol	C6H12O6	0.48
10	15.594	d-mannose pyran	C16H32O2	3.29
14	17.396	Palmitic acid	C18H36O2	0.85
19	21.202	Eighteen carbonate	C15H14O6	5.08
21	21.779	Epicatechin	C15H14O6	2.09
23	24.034	Epicatechin	C15H14O6	7.31
24	24.492	Epicatechin	C15H14O6	7.74

FUNDING

This work was supported by “The Fundamental Research Funds for the Central Universities of China” (No. BLYJ201203), and The National Natural Science Funds of China” (No. 31271981).