Speciation and determination of inorganic antimony in tea infusion by using solid-phase extraction-based liquid chromatography-inductively coupled plasma-mass spectrometry

Abstract In this work, we developed a method to separate and detect inorganic antimony (iSb) in tea infusions based on liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS). Considering that iSb was present in tea infusions in trace amounts, a commercial solid-phase extraction (SPE) column was employed to enrich iSb(III) and iSb(V). In this study, we found that iSb exists in tea infusions in a bound state with an unknown substance rather than in a free state. Therefore, the enrichment step was combined with oxidation and reduction processes. Because of the enrichment process and the high sensitivity of LC-ICP-MS, the LODs of iSb(III) and iSb(V) were both as low as 0.03 μg L−1. The RSDs (n = 6) of iSb(III) and iSb(V) were all below 8.1%, and the recoveries (n = 6) were 92–96% and 90–94%, respectively. Then, a total of six tea infusion samples were inspected. The data revealed that the total Sb in the tea infusions ranged from 0.22 to 0.97 μg L−1, and we found that 50–83% of the total Sb in the tea infusion was iSb. This method is worth promoting since it has satisfying methodological performance. Graphical abstract


Introduction
As a popular beverage worldwide, tea has been readily consumed for thousands of years with enormous consumption [1,2].Studies have revealed that the enriched metal and metalloid elements [3,4] in tea can be partially dissolved into a tea infusion during soaking, leading to health risks with consumption.Among these elements, antimony is highly toxic and deserves further attention [5].Sb compounds can be divided into inorganic Sb and organic Sb, in which the toxicity of inorganic Sb is much higher than that of organic Sb [6].Therefore, it is of great importance to study iSb in tea infusions and quantitatively analyze it as well.It is commonly known that iSb has two forms, iSb(III) and iSb(V), which can be easily converted into each other.In oxidizing environments, such as on the surface of a river and in the air, the predominant form of iSb is iSb(V), in contrast, in reducing environments, such as in plant roots, iSb(III) is the major species [7][8][9].Despite the high toxicity of iSb and the high consumption of tea, studies focused on the speciation and determination of iSb in tea diffusion are rare due to technological obstacles.The main hindrance is that the total Sb in tea is present at trace levels [3], so the amount of iSb should be relatively low.Reports revealed that the methods for antimony enrichment were as follows: supramolecular solvent-based liquid phase extraction [10], BPHA-[C 4 mim][PF 6 ] single drop microextraction [11], air-assisted liquid-liquid microextraction (AA-LLME) [12], narrow-bore tube dispersive liquid-liquid microextraction (NT-DLLME) [12], polymer monolithic capillary microextraction [13], and ultrasound-assisted magnetic solid phase extraction (UA-MSPE) [14].However, most of these pretreatment methods are complicated, time-consuming and have never been applied in a tea infusion matrix.With the help of a new synthetic enrichment material, Chen et al. [15] detected iSb in tea and its infusion by using electrothermal vaporization-inductively coupled plasma-mass spectrometry (ETV-ICP-MS).Their study proved that Sb and iSb were present in the tea infusion at ultralow concentrations.However, their new synthetic enrichment material was not a commercial consumable material, which limited further popularization of the method.Therefore, developing a common enrichment and detection method for iSb determination in tea infusion is meaningful.
LC-ICP-MS, a powerful speciation tool, has been widely used in the analysis of food, drinking water, biological samples, and environmental samples, covering many metal and metalloid element studies [16][17][18][19][20][21][22].In Sb speciation studies using LC-ICP-MS, researchers obtained extremely high sensitivity, and the LODs of iSb(III) and iSb(V) were as low as 0.03-3.2lg L À1 and 0.01-1.0lg L À1 , respectively [23,24].Despite great progress in Sb speciation, two difficulties have greatly affected the quantitative analysis of trace iSb.One was that the ultralow trace level of iSb in some samples was lower than the method's LOD, and another was the inferior peak shape of iSb(III), compromising the accuracy of its peak area integration.Therefore, iSb determination in tea infusion must overcome these two difficulties.SPE-based enrichment processes are widely employed in many trace analyses to improve the sensitivities of the method and remove interference [25,26].In addition, transforming iSb(III) into iSb(V) can not only obtain better chromatographic performance for peak area integration but also shorten the analysis time on LC-ICP-MS.These two pretreatment skills were obviously suitable for iSb determination in this study.
In the present work, we found that iSb exists in tea infusion as a binding state with an unknown substance rather than as a free state.This has not been reported before.To analyze the trace iSb level in the tea infusions, optimized pretreatment and detection steps were carried out as follows: First, the tea infusion was passed through the SPE column to enrich iSb(III) and leave iSb(V) in the filtrate.After rinsing the SPE column with purified water, elution of the SPE cartridge with the oxidating solution so the enriched iSb(III) could be instantly oxidized into free-iSb(V) and eluted for further quantitative analysis via LC-ICP-MS.Addition of a reduction agent in the previous step's eluate was made, to achieve transformation of iSb(V) to iSb(III).The SPE procedure was then repeated for concentration of iSb(III) on the absorbent and for elution in oxidative conditions under iSb(V) form.With this method, iSb(III) and iSb(V) could be successfully detected.
This method is worth promoting since it not only has satisfying methodological performance but also only uses commercial reagents and materials that could be easily purchased.Furthermore, in real sample analysis, data revealed that the major form of Sb in tea infusion was iSb, and within iSb species, the concentration of iSb(III) was generally higher than that of iSb(V), indicating that the risk of Sb intake from tea infusion was primarily from iSb, especially iSb(III).

Material
EDTA-Na 2 (30 mM, Nanjing Chemical Reagent Factory, Nanjing, China) was applied to prepare the mobile phase.Vitamin C and L-cysteine (Nanjing Chemical Reagent Factory, Nanjing, China) dissolved in diluted hydrochloric acid (Merck, MO, USA) were used to reduce iSb(V) to iSb(III) in tea infusion.SbCl 3 and potassium pyroantimonate (Merck, MO, USA) were purchased to prepare iSb(III) and iSb(V) standard solutions.The oxidation process to iSb(III) in tea infusion was conducted by using diluted H 2 O 2 þ NaOH (Nanjing Chemical Reagent Factory, Nanjing, China) mixed solution as the oxidizing reagent.Antimony and rhodium standard solutions (Spex, NJ, USA) diluted with 1% (v/v) HNO 3 (Merck, MO, USA) were prepared for total Sb determination; 60 mg 3 mL MAX, WAX, MCX, MAX, HLB (Oasis, MA, USA) SPE columns were compared in the pretreatment stage, and these SPE columns were all activated followed by their instructions before use.Pure water was produced by a Milli-Q integral water purification system (Merck, Molsheim, France).

Equipment and parameters
Tea leaves were ground to powder in a crusher (Midea, Guangzhou, China) before soaking.After soaking, the tea infusion was separated from the residue by centrifugation (3-18, Sigma, Osterode, Germany).An L-20AB liquid chromatograph (Shimadzu, Japan) equipped with an anion exchange column (Hamilton PRP X100, 5 mm, 250 mm Â 4.6 mm, NV, USA) connected to a 7900 ICP-MS (Agilent, CA, USA) was the main equipment for iSb separation and determination.The optimized conditions are shown in Supplementary Table 1.

Sample pretreatment
Soaking period of the tea infusion A total of three black tea samples (No.1: Dianhong Jinya, Yunnan Province; No.2: Jinjunmei, Fujian Province; No.3: Qimen Black tea, Anhui Province) and three green tea samples (No.4: Maofeng Greentea, Sichuan Province; No.5: Maoshan Cloud-fog Tea, Jiangsu Province; No.6: Longjing, Zhejiang Province) were purchased from the local market and then ground into tea powder.A total of 5.0 g tea powder was placed into a 500 mL beaker, soaked with 250 mL boiling pure water and cooled at room temperature for 5, 10, 15, and 20 min to obtain tea infusion.Before analysis, the tea infusion samples were centrifuged at 7000 r/min for 10 min to remove solid residues.

Determination of total Sb in the tea infusion
The total Sb in tea infusion was directly detected by ICP-MS with 10.0 lg L À1 Rh as the online internal standard.A total of 5.0 lL of 1000 lg L À1 Sb standard solution was added to 10 mL tea infusion to make the spiked samples.

Determination of iSb in the tea infusion
The steps for the quantitative determination of iSb in the tea infusion were as follows: First, 10 mL of the tea infusion was passed through the HLB SPE column to enrich iSb(III) and leave iSb(V) in the filtrate.The SPE column was rinsed with 2 mL of purified water and then eluted with 1.0 mL of 2% (v/v) H 2 O 2 þ1% (w/v) NaOH.At this point, the 10-fold enriched iSb(III) in the SPE column could be instantly oxidized into free iSb(V) and eluted for LC-ICP-MS analysis.After collection of the filtrate (approximately 9.6 mL) from the previous step, the addition of 0.1 mL HCl þ 0.05 g L-cysteine was made, followed by a waiting time of 5 min to achieve complete reduction of iSb(V) to iSb(III).The pH value of the eluate was previously fixed at 5.5-7.0 (the pH of the original tea infusion).Then, all the filtrate was loaded onto the HLB SPE column to enrich iSb(III).The SPE column was rinsed with 2 mL of pure water and then eluted with 1.0 mL of 3% (v/v) H 2 O 2 þ1% (w/v) NaOH.Here, iSb(III) could be oxidized back to iSb(V) for further LC-ICP-MS analysis.Finally, the iSb concentration could be obtained by adding the amounts of iSb(III) and iSb(V).A total of 10.0 lL of 500lg L À1 iSb(III) or iSb(V) was added to a 10 mL tea infusion to make the respective spiked samples.

Method validation
Total Sb determination: For the total Sb in the tea infusion in trace amounts, the calibration curve was set by diluting the Sb standard (1000 mg L À1 ) to 0, 0.1, 0.5, 1.0, 1.5, and 2.00 lg L À1 .The 10 lg L À1 Rh solution as the online internal standard was simultaneously pumped into the ICP-MS throughout the analysis.The RSD (n ¼ 6) was obtained by analyzing the total antimony of sample No. 2 repeatedly, and the recoveries (n ¼ 6) were acquired by spiking 0.5 lg L À1 Sb standard in sample No. 2. A 0.1 lg L À1 Sb standard solution was measured 11 times to obtain the standard deviation (SD), and then the LOD was calculated as 3SD/slope [27].iSb(III)or iSb(V) determination: The calibration curve of iSb(III) or iSb(V) was obtained by diluting the SbCl3 or potassium pyroantimonate solution to 0, 0.1, 0.2, 0.5, 0.8, and 1.0 lg L À1 .The standards went through the whole pretreatment process before LC-ICP-MS analysis.The RSD (n ¼ 6) was obtained by analyzing iSb(III) or iSb(V) of sample No. 2 repeatedly, and the recoveries (n ¼ 6) were acquired by spiking 0.5 lg L À1 iSb(III) or iSb(V) solution in sample No.2.A 0.1 lg L À1 iSb(III) or iSb(V) solution was measured 11 times to obtain the standard deviation (SD), and then the LOD was calculated as 3SD/slope [27].

Determination of total Sb in the tea infusion
It is more relevant to determine the total Sb content in tea infusions than in tea leaves because not all Sb in tea leaves is leached out during tea brewing [15].Studies [3,4] revealed that the soaking time affected the metal and metalloid species dissolution in tea, so the Sb species should exhibit the same behavior.In the present work, the soaking time influence was examined, as shown in Figure 1.It should be noted that samples No. 1-No. 3 were black tea, and samples No. 4-No.6 were green tea.
In Figure 1, the total Sb content in black tea infusion and in green tea infusion ranged from 0.28-0.97lg L À1 and 0.22-0.46lg L À1 , respectively.Approximately 70-83% soluble Sb was dissolved in the first 5 min, and the content reached equilibrium after 15 min, indicating that the dissolution of soluble Sb mainly occurred in the early stage of soaking.Since there is no Sb concentration limit for tea infusion, here we quoted the Brazil food standard for Sb in fruit juices( 1.0 lg L À1 ) [28], and the WHO limit for total Sb in drinking water ( 5 lg L À1 ) [29] to roughly evaluate the health risk of these tea infusion samples.The risks of total Sb in these tea infusion samples were low.

The forms of iSb in the tea infusion
Before investigating the species of iSb, a method based on LC-ICP-MS for the separation of iSb(III) and iSb(V) should be established.iSb speciation methods have been widely discussed before.However, among these methods, the chromatographic behavior of iSb(III) was usually inferior to that of iSb(V), as the retention time was longer and the peak shape was worse.In this study, the speciation of iSb was carried out, as shown in Figure 2.
Figure 2 shows that the iSb(III) and iSb(V) retention times were 7.7 min and 2.6 min, respectively.The peak shape of iSb(III) was of low height and had a long tail, and it was difficult to calculate its peak area when its concentration was low.The concentration of iSb(III) in tea infusion should be low since the total Sb was in trace amounts (Figure 1).As a result, direct determination of iSb(III) on LC-ICP-MS was not suitable.Furthermore, free iSb (both free iSb(III) and free iSb(V)) did not exist in all tea infusion samples, which increased the difficulty of iSb determination, as shown in Figure 3.
Compared to the standard chromatograms of iSb in Figure 2, no free iSb(III) or free iSb(V) was found in the tea infusion (Figure 3a).The spike experiment further showed that iSb(III) easily bound to some substance in the matrix in the tea infusion (Figure 3b), and the free iSb(III) (Figure 3b) disappeared in the chromatogram after 5 min (Figure 3c).Similarly, iSb(V) also bonded to some substance in the matrix, such as Sb-1 and Sb-2 (Figure 3d), and the amount of spiked free iSb(V) gradually decreased over time (Figure 3e), and no free iSb(V) remained in the system when the incubation time reached 20 min.Unlike the iSb(III)-matrix complex, the predominant form of the iSb(V)-matrix complex was Sb-1 (t ¼ 2.8 min), which could be eluted under the applied chromatographic conditions.The retention times of Sb-1 and free iSb(V) were close, and their peak shapes were similar, suggesting that they had similar chromatographic characteristics in the anion exchange column.Since the chromatographic characteristics of the main peak in Figure 3a were coincident with Sb-1, Sb-1 as the main form of iSb(V) in tea infusion was obvious.Thus, the conclusion that no free iSb(V) existed in the tea infusion was confirmed.
To investigate the difference in intensities per unit between Sb-1 and iSb(V) in ICP-MS, 20.0 lg L À1 of iSb(V) was spiked into the tea infusion No. 2 and stabilized for 20 min (to ensure that all iSb(V) was changed to Sb-1).Then, the spiked tea infusion sample was injected into the LC system to separate and collect the eluent between retention times of 2.0-4.0 min.Therefore, 2.0 mL of eluent containing Sb-1 was collected.The above eluent was concentrated to approximately 0.2 mL by using a boiling water bath.Then, 0.3 mL of HNO 3 was added into the system to digest for 2 hr and diluted to 2 mL.After digesting, the total Sb in Sb-1 was determined by ICP-MS.With the same LC and digestion procedures, 20.0 lg L À1 iSb(V) standard solution was also inspected.The results showed that the mean values of total Sb in Sb-1 and in the iSb(V) standard solution were 0.463 lg L À1 (n ¼ 3) and 0.480 lg L À1 (n ¼ 3), respectively.This indicated that the intensities per unit of iSb-1 and iSb(V) were almost the same in ICP-MS.

Determination of iSb in the tea infusion
To accurately determine iSb on LC-ICP-MS, it is essential to release free iSb from the binding substance in the matrix and enrich iSb.Here, we carried out a scheme as follows.First, we employed the proper SPE column to enrich the iSb(III) complex in the tea infusion while leaving the iSb(V) complex in the filtrate.The SPE column was eluted with the oxidizing eluent to oxidize the iSb(III) complex to obtain free iSb(V) online during elution.Thus, the enriched iSb(III) could be quantitatively detected in the form of iSb(V) on LC-ICP-MS.Second, a suitable reducing reagent was chosen to reduce the iSb(V) complex to iSb(III), the iSb(III) complex was enriched by the SPE column, and the previous oxidizing wash-out process was repeated to obtain enriched iSb(V) for LC-ICP-MS analysis.Finally, as the amounts of iSb(III) and iSb(V) were both measured, the iSb concentration in the tea infusion was subsequently determined.In the scheme, three key points mattered a lot: the selection of the oxidizing eluent, the choice of the SPE column, and the optimization of the reduction reagent.
Selection of the oxidizing eluent: To seek a proper oxidizing eluent, 0.5 lg L À1 iSb(III) was spiked in the sample No. 2 tea infusion and incubated for 5 min.The HLB SPE column was used to filter 10 mL of the spiked tea infusion to enrich iSb(III), and then the SPE column was rinsed with 2 mL of pure water.Note that the HLB SPE column did not retain iSb(V).One milliliter of alkaline oxidation elution at different concentrations was injected into the SPE column to oxidize iSb(III) to free iSb(V).The eluate containing free iSb(V) was analyzed by LC-ICP-MS, and as a result, iSb(III) was indirectly measured.The data are shown in Supplementary Table 2.We discovered that the more H 2 O 2 and NaOH added, the higher the oxidation efficiency of the eluent.When the concentrations of H 2 O 2 and NaOH in the eluent were greater than 2% (v/v) and 1%(w/v) respectively, the recoveries reached a plateau (>90%).Therefore, 2% (v/ v) H 2 O 2 þ 1% (w/v) NaOH solution as the oxidizing eluent was employed in this study.The behavior of the iSb(III) complex could be complicated in tea infusions, and some of the iSb(III) species could not be enriched or oxidized under  the given conditions.Fortunately, the spiking experiment indicated that most (94%) of the iSb(III) complex could be oxidized and washed out, which was satisfactory.
Selection of SPE columns: Due to the inability to confirm the morphology of iSb(III) and iSb(V) complexes prior to determination, we screened solid-phase extraction columns with different performances, including WCX, MCX, WAX, MAX, and HLB.The specific steps are shown as follows: 5.0 lg L À1 iSb(V) was spiked in the sample No. 2 tea infusion and incubated for 20 min.All the SPE columns were first washed with 5 mL 0.1 g L À1 vitamin C and then rinsed with pure water before use to remove residual oxidation in the SPE columns.The spiked tea infusion was then passed through these SPE columns with a loading volume of 10 mL.The chromatograms of Sb species in the filtrate are shown in Supplementary Figure 4.
Measuring the peak area of the chromatograms in Supplementary Figure 4, it could be seen that the MCX (Supplementary Figure 4b) and WAX (Supplementary Figure 4c) SPE columns could partially retain iSb(V), and unlike the chromatograms of the MAX (Supplementary Figure 4d) and HLB (Supplementary Figure 4e) filtrates, the iSb(V) form after filtering through the WCX (Supplementary Figure 4a) column was not free iSb(V) but iSb-1.Therefore, MAX and HLB SPE columns were preferred in this section.The enrichment efficiency of each SPE column to iSb(III) was also evaluated via the spiking experiment.After enrichment and elution with 1 mL of 2% (v/v) H 2 O 2 þ1% (w/v) NaOH, the analyte was measured by LC-ICP-MS.The results are shown in Supplementary Table 3 (n ¼ 3).The data in Supplementary Table 3 revealed that the WAX and MAX SPE columns could not collect iSb(III) efficiently since only <30% analyte was found.The MCX SPE column was much better but could still not meet the requirements of the method, as its average recovery was 62%.Luckily, the WCX and HLB SPE columns performed well, and their average recoveries were 92% and 94%, respectively.Combined with the previous work (seen in Supplementary Figure 4), the HLB SPE column was the best choice in this study.
Selection of the reduction reagent: Although iSb(V) in tea infusion of sample No.2 could be measured directly on LC-ICP-MS after HLB SPE column purification, iSb(V) in most samples is at a trace level, such as No. 5 and No. 6 tea infusion samples, which could not be accurately analyzed without enrichment.In this study, iSb(V) was proposed to reduce to iSb(III) before enrichment.Different reduction systems were applied and compared by spiking 5.0 lg L À1 iSb(V) into the tea infusion of sample No.2, as shown in Supplementary Figure 5.
In Supplementary Figure 5, compared with the reduction efficiency of vitamin C (Supplementary Figure 5a), L-cysteine (Supplementary Figure 5b,c) had a much better performance.When 0.5% (w/v) L-cysteine (Supplementary Figure 5b,c) was applied, 1% (v/v) HCl (Supplementary Figure 5c) had a more thorough reduction performance than 0.5% (v/v) HCl (Supplementary Figure 5b) and met the experimental requirements.
Hence, the enrichment process of iSb(V) was summarized as follows: 10 mL filtrate of the tea infusion was collected after HLB SPE column filtering, and 0.1 mL HCl þ 0.05 g L-cysteine was added into the filtrate and incubated for 5 min to reduce iSb(V) to iSb(III) completely.The pH value of the above acid solution was neutralized to 5.5-7.0.Then, all the solutions were loaded onto the HLB SPE column to enrich iSb(III).The SPE column was rinsed with 2 mL of pure water, and then eluted with 1.0 mL of 3% (v/v) H 2 O 2 þ1% (w/v) NaOH.Unlike the usage of 2% (v/v) H 2 O 2 þ 1% (w/v) NaOH in the previous section, 3% (v/v) H 2 O 2 þ 1% (w/v) NaOH was applied here for elution since excess H 2 O 2 could oxidize residual L-cysteine in the HLB SPE column.A spiking experiment was conducted by adding 0.5 lg L À1 iSb(V) to the tea infusion of sample No.2 (n ¼ 3), and the average recovery was determined to be 92%.

Method validation
Following the steps described in "Determination of iSb in the tea infusion", we studied the figures of merit of the method, as shown in Supplementary Table 4.
As shown in Supplementary Table 4, we found that when determining the total Sb content in the tea infusion, the LOD could be as low as 0.09 lg L À1 , which was suitable for ICP-MS determination of Sb under such conditions.The RSD was 5.6%, and the recoveries were 97%-102%.
Compared to the total Sb detection by using ICP-MS, the sensitivity of Sb should decline in LC-ICP-MS analysis since the loading volume in LC-ICP-MS was less than that of in ICP-MS.However, when SPE-based enrichment skills were applied, the sensitivities of Sb species could be subsequently increased.As iSb(III) was converted to iSb(V) before LC-ICP-MS analysis, the better chromatographic behavior of iSb(V) could also promote the sensitivity of iSb(III) determination.As a result, the LODs of iSb(III) and iSb(V) were both 0.03 lg L À1 .The recoveries of iSb(III) and iSb(V) were 92-96% and 90-94%, respectively, and their RSDs were all below 8.1%.These validation data suggested that the method could meet the requirement of trace iSb determination in tea infusion.

Real sample analysis
Samples were analyzed at different soaking times, as shown in Tables 1-3.
The data in Tables 1-3 revealed that 50-83% of the total Sb in tea infusion was iSb, the ratio was relatively stable during soaking, and no significant difference in the ratio was observed between black tea and green tea.Furthermore, the amount of iSb(III) was generally higher than that of iSb(V) in tea infusion, as the concentrations of iSb(III) and iSb(V) were 0.08-0.39lg L À1 and 0.05-0.34lg L À1 respectively, and the relative ratio of ciSb(III): ciSb(V) was 0.88-3.2.
The results indicated that the uptake risk from Sb in the tea infusion was mainly in the iSb form, particularly iSb(III).

Conclusions
In the present work, a method based on LC-ICP-MS was established to detect trace amounts of iSb in tea infusions.For the first time, we proved that there iSb is not present in tea infusions in free form, and iSb is conjugated with some substance in the matrix.To quantitatively analyze iSb, the HLB SPE column combined with oxidizing and reducing processes was employed to convert and enrich the analyte.The LODs of iSb(III) and iSb(V) were both as low as 0.03 lg L À1 , which was sufficient for real sample analysis.Moreover, a total of six tea samples, including black tea and green tea were analyzed.The results showed that the dissolution of soluble total Sb in tea mainly occurred in the early stage of soaking.Approximately 50-83% of the total Sb was iSb, and the amount of iSb(III) was generally higher than that of iSb(V) in the tea infusion, as the ratio of ciSb(III): ciSb(V) was 0.88-3.2,indicating that iSb(III) was the main Sb species in the tea infusion, which is an indication of a health risk.Unfortunately, there is no limit regulation for iSb in tea infusion, so the health risk of iSb was difficult to evaluate.In addition, only commercial reagents and consumables were applied in this method, and such low LODs of iSb were obtained, suggesting that the method could be easily implemented in laboratories for iSb detection not only in tea infusion samples but also in other liquid samples of a similar matrix.

Figure 1 .
Figure 1.Total Sb in tea infusion samples at different soaking time.

Table 1 .
Determination of iSb(III) in tea infusions at different soaking time (n ¼ 3).

Table 2 .
Determination of iSb(V) in tea infusions at different soaking time (n ¼ 3).

Table 3 .
Determination of iSb/Total in tea infusions at different soaking time (n ¼ 3).