Analysis of Phthalimide in Tea Based on Derivatization with Isoamyl Bromide and Gas Chromatography-Mass Spectrometry

Abstract

Phthalimide can be formed through the degradation of folpets and phosmet or the reaction of phthalic anhydride with ammonia. The sum of phthalimide and folpet is expressed as the folpet residue. Thus, an analytical method is urgently required to investigate phthalimide residue levels and sources in tea. Herein, we developed a new method for determining phthalimide content using acetone extraction and isoamyl bromide derivatization coupled with gas chromatography-mass spectrometry, which had satisfactory precision and accuracy, and was used to analyze 337 green tea, 36 oolong tea, and 91 black tea samples. Phthalimide was detected in most samples. The maximum level of phthalimide was 16.2 μg/kg, 12.6 μg/kg, and 11.8 μg/kg for green tea, oolong tea, and black tea, respectively. There was no statistically significant difference across tea types. Our observations provide crucial information and an analytical method to further study the sources and related pathways of phthalimide formation in tea.

Introduction

Phthalimide can be formed from the degradation of the fungicide folpet and insecticide phosmet or the reaction of phthalic anhydride with ammonia (EFSA, 2017; Jones et al., 2021). As a contact fungicide, folpet is extensively used on various crops to prevent leaf diseases. In Japan, folpet is registered as a pesticide for adzuki beans, cucumbers, tomatoes, melons, and onionsⁱ⁾. The insecticide phosmet has not yet been registered in Japan. According to the European Food Safety Authority (EFSA), folpet residues in crops are almost completely converted to phthalimide under high temperatures, alkaline pH, and analytical procedures in sample preparation (EFSA, 2013). Accordingly, the European Union (2016) set a new residue definition: the sum of folpet and phthalimide levels, which is expressed as the folpet residue.

  Tea has multiple biological activities, making it one of the most popular non-alcoholic beverages worldwide. Tea is not only consumed as a beverage but also as a powder for use in various food items. During tea manufacturing, tea leaves are dried in hot air at approximately 100 °C. In particular, green tea is heated between 100 °C and 180 °C at the end of the manufacturing process using a roaster. At high temperatures, the folpet is completely degraded and converted into phthalimide (Han et al., 2022). Tea leaves contain a significant amount of phthalic anhydride (Wittig et al., 2022), which can easily react with ammonia. Therefore, phthalic anhydride in tea leaves can form phthalimides during tea manufacturing (Gao et al., 2019). To evaluate both levels of folpet residues and phthalimide formation, an analytical method is urgently needed to investigate folpet residue levels and the sources of phthalimide in tea.

  Analytical methods based on gas chromatography (GC) and/or liquid chromatography (LC) coupled with mass spectrometry (MS) have been used to quantify phthalimide in foodstuffs (Huertas-Pérez et al., 2018; Huertas-Pérez et al., 2019; Gao et al., 2019; Han et al., 2021; Sun et al., 2021; and Wittig et al., 2022). These studies used analytical equipment such as GC-MS/MS and LC-MS/MS for phthalimide quantification. Furthermore, high-resolution MS can be used for the analysis of phthalimide. However, to the best of our knowledge, there have been no reports of phthalimide analysis using GC coupled with a simple quadrupole mass spectrometer, which is widely used in many laboratories. Moreover, there have been cases where phthalimide was analyzed using a flame ion detector (Menzio et al., 2021); however, quantifying low levels of phthalimide in tea samples remains difficult.

Fig. 1.

Phthalimide derivatization with isoamyl bromide for gas chromatography-mass spectrometry analysis.

In this study, we established a method for the quantification of phthalimide in tea via derivatization with isoamyl bromide followed by GC-MS. Validation was performed according to the SANTE/11312/2021 guidelines (European Commission, 2021). Furthermore, the phthalimide levels in tea samples made in Japan and sold in markets were determined. This study provided crucial information and reported an analytical method for the further study of the sources and related pathways of phthalimide formation in tea.

Materials and Methods

Chemicals　　Before use, acetonitrile, acetone, and hexane (Fujifilm Wako Pure Chemical Co., Ltd.) were distilled using a three-ball Snyder column. The distilled solvents were stored in glass Erlenmeyer flasks under an argon atmosphere. Phthalimide, isoamyl bromide, and potassium carbonate were obtained from Tokyo Chemical Industry Co., Ltd. Isoamyl bromide was distilled using short-path distillation equipment. Potassium carbonate was washed with distilled acetone prior to use. Phthalimide-d4 (99.0 % purity) was obtained from CDN Isotopes (Pointe Claire, Quebec, Canada). Anhydrous sodium sulfate was purchased from Fujifilm Wako Pure Chemical Co., Ltd.

Tea samples　　Green (n = 337), oolong (n = 36), and black (n = 91) tea samples were purchased from local markets and tea traders in Japan between November 2022 and December 2024. In the present study, no phthalimide-free tea samples were found. Therefore, the tea samples with the lowest levels of phthalimide for each type of tea were used for analytical method validation.

Preparation of tea samples　　To prevent artifact contamination, all glassware and perfluoroalkoxyl alkane (PFA) tubes used for extraction before derivatization with isoamyl bromide were washed with distilled acetone. For phthalimide extraction, tea samples were frozen in liquid nitrogen and pulverized using a cyclone sample mill (3010-018, Udy Corp., Fort Collins, CO, USA). The ground tea powder (1.00 g) was weighed in a 50 mL PFA centrifuge tube. Phthalimide-d4 and distilled acetone (40 mL) were added to the ground tea powder in PFA tubes. The tea samples with the lowest level of phthalimide for each type of tea were used for the recovery test.

A phthalimide standard solution was added to each tea sample to increase its content by 0.65, 1.30, 2.60, 10.4, and 20.8 μg/kg. For phthalimide extraction, the mixture was shaken vigorously at 1 500 rpm for 30 min (High-shaker, CM-1000, Tokyo Rikakikai Co., Ltd.). The mixture was then centrifuged at 5 000 rpm for 10 min. Finally, 15 mL of the supernatant was transferred to a PFA vial with a screw cap for subsequent derivatization.

Derivatization reaction with isoamyl bromide　　After extraction, potassium carbonate (0.2 g) was added to the supernatant (15 mL), followed by the addition of distilled isoamyl bromide (0.20 mL). A stir bar was placed in a PFA vial, and the vial was closed with a screw cap. The reaction solution was stirred for 1 h at 60 °C (Fig. 1).

Clean-up procedure　　After cooling, the solution was evaporated to dryness using a rotary evaporator at 40 °C and redissolved in acetonitrile (4 mL). The solution was applied to a solid phase extraction (SPE) cartridge (InertSep C18 500 mg/3 mL, GL Sciences Inc.) preconditioned with 2 mL of acetonitrile. The SPE eluate was evaporated to dryness using a rotary evaporator at 40 °C and redissolved in hexane (2 mL). The solution was then applied to an SPE cartridge (InertSep GC, 50 mg/mL, GL Sciences Inc.) containing 2 mL of hexane. The eluate was evaporated to approximately 0.5 mL using a rotary evaporator at 40 °C. Finally, the sample was concentrated to 0.05 mL using a nitrogen stream before GC-MS analysis. Phthalimide was extracted from tea powder according to a previously described method, and the color of the extract was clarified via purification using SPE cartridges after derivatization with isoamyl bromide. This should reduce the risk of contamination of analytical equipment.

Instrumentation conditions　　To identify the phthalimide derivatives, mass spectra were generated using a mass spectrometer (JMS-Q1500GC; Jeol) in the electron impact ionization mode at 70 eV. The source and transfer line temperatures were set to 230 °C. A scan range of m/z 30–400 at a rate of 2.05 scans/s was employed. To quantify N-isoamylphthalimide, the extracted ion peak area was obtained using mass chromatography in the selected ion mode. The extracted ions were monitored at m/z 160 and m/z 217 for N-isoamylphthalimide, m/z 164 and m/z 221 for N-isoamylphthalimide-d4 as an isotopically labeled internal standard. A fused-silica capillary column, DB-225MS (30 m × 0.25 mm i.d., 0.25 μm film thickness; J & W Scientific, Folsom, CA, USA), was used to identify and quantify N-isoamylphthalimide. Helium (>99.999 % purity) was used as the carrier gas at a constant flow rate of 1.0 mL/min. The concentrate (1 μL) was injected into the column in the splitless injection mode, and the injector temperature was maintained at 230 °C. The oven temperature was maintained at 40 °C for 1 min, increased at a rate of 40 °C/min to 200 °C, maintained at 200 °C for 5 min, increased again at a rate of 40 °C/min to 230 °C, and maintained at 230 °C for 15 min.

Method validation　　The validity of the quantitative analysis of phthalimide in tea samples was evaluated according to the SANTE/11312/2021 guidelines based on the linearity, additive recovery rate, repeatability, intermediate precision, robustness, and limit of quantification (LOQ). Linearity was evaluated by analyzing phthalimide standard solutions. A calibration curve was constructed for quantification using isotopic dilution. The measurements were performed in triplicate by using five phthalimide standard solutions with different concentrations (0.10, 0.40, 0.77, 0.96, and 1.85 μg/L). The isotopic internal standard was spiked at a concentration of 2.03 μg/L and processed according to the sample preparation, derivatization reaction, and clean-up procedure described previously. The relative peak area of each analyte (native/isotopically labeled) was plotted against its concentration ratio (native/isotopically labeled). The calibration curve within the selected working range was evaluated using the plotted data (area ratio and concentration ratio of the analyte) and the correlation coefficient (r). In addition, P-values for the lack-of-fit test at a 95 % confidence interval (α = 0.05) were obtained using Microsoft Excel (Microsoft 365 MSO).

The commercially available teas (green tea, oolong tea, and black tea) with the lowest levels of phthalimide detected were used for the recovery test, as phthalimide-free tea samples were not available. The lowest phthalimide level was 0.64 μg/kg, 2.97 μg/kg, and 2.05 μg/kg in green tea, oolong tea, and black tea, respectively. Phthalimide standard solutions were added to each tea sample to increase its phthalimide content by 0.65, 1.30, 2.60, 10.4, or 20.8 μg/kg. The additive recovery rate was evaluated by analyzing each tea sample with the lowest level of phthalimide spiked with the standard solutions. For the recovery tests, five parallel extractions were performed daily at the same spiking level. Then, five spiked extracts were analyzed on five successive days. To evaluate repeatability and intermediate precision, one-way analysis of variance (ANOVA) was performed using Microsoft Excel (Microsoft 365 MSO) to obtain both within-day and inter-day variances. The relative standard deviations (RSDs) were then calculated:

  

・・・・・・ Eq. 1   

・・・・・・ Eq. 2

where RSD_rep is the RSD of repeatability (%), RSD_ip is the RSD of intermediate precision (%), the difference variance is the difference between the inter- and within-day variance obtained from the one-way ANOVA, mean recovery is the mean recovery rate in all observations (n = 25), and n is the number of observations in each day (n = 5).

  Robustness is defined as the mean recovery rate for all observations (n = 25). The LOQ was estimated based on the minimum concentration that provided an adequate recovery range (70–120 %) and repeatability value (RSD ≤ 20 %).

Results and Discussion

Phthalimide derivatives　　Chemical derivatization is frequently used to enhance the signal intensity for GC analysis. In our preliminary experiment, phthalimide was not detected in tea samples using GC-MS. Therefore, phthalimide should be derivatized to enhance its signal intensity and to quantify it. To enhance the intensity of phthalimide, 3-bromopropyltrimethylammonium bromide (BPTAB) coupled with ultra-high performance LC and high-resolution MS (Gao et al., 2019) was used for derivatization. However, in the case of GC analysis, the elution time of BPTAB appeared to be delayed due to its low volatility, which was not suitable for conventional quantitative analysis. Therefore, another derivatization reagent must be used for GC analysis. Therefore, we investigated a method for the derivatization of phthalimide for GC analysis. To develop a subtle method for phthalimide derivatization, phthalimide was derivatized using various alkyl bromides. In our preliminary studies, methyl, ethyl, propyl, butyl, amyl, hexyl, and isobutyl phthalimides were detected in the green, oolong, and black tea samples. However, isoamyl phthalimide was not detected in any tea sample. Therefore, isoamyl bromide was used to derivatize phthalimide for GC analysis. Phthalimide alkyl derivatives can be formed from phthalic anhydrides and alkyl amines (Paula et al., 2022). Tea leaves contain phthalic anhydride (Witig et al., 2022) and alkyl amines, including methyl, ethyl, propyl, butyl, amyl, and hexyl amines (Tsushida, 1987). In our preliminary study, fresh tea leaves did not contain phthalimide alkyl derivatives before processing. Therefore, phthalic anhydride in tea leaves can react with alkylamines during tea manufacturing to produce phthalimide alkyl derivatives. Fig. 2 shows the mass spectra of N-isoamylphthalimide and N-isoamylphthalimide-d4. The most intense product ion of N-isoamylphthalimide had an m/z 160, and its molecular ion had an m/z 217. The most intense product ion of the stable isotope (d4) had an m/z 164, and its molecular ion had an m/z 221.

Fig. 2.

Mass spectra of N -isoamylphthalimide and N -isoamylphthalimide- d4 .

Blank test　　At the beginning of establishing the method, we conducted a blank test. Extraction, purification using SPEs, and derivatization were performed as described in the Materials and Methods section, except that phthalimide-d4 and tea powder were not added. The peaks derived from the solvents, reagents, and equipment used for extraction, refinement, and derivatization had the same retention times as the peaks of isoamyl phthalimide at m/z 160 and m/z 217. Some research groups have already shown that the presence of phthalimide is due to artifact formation during the analysis process (Huertas-Pérez et al., 2018; Huertas-Pérez et al., 2019; Relana, 2017). The potential generation of phthalimide from sources different than folpet breakdown, e.g., the reaction of phthalic anhydride and a nitrogen source, can occur (EFSA, 2017; Jones et al., 2021; Relana, 2017). Phthalic anhydride is a ubiquitous compound used in resins, paintings, plastics, and other applications, and is known to react with urea or ammonia at high temperatures (Relana, 2017). This reaction could occur during analysis, such as during sample preparation or GC hot injection. Isoamyl phthalimide was detected in the blank test; hence, it was suspected to be a contamination from the solvent, reagents, glassware, and PFA tubes used before derivatization. To avoid contamination, acetone was used as the extraction solvent, which was distilled using a three-ball Snyder concentrator. The distilled solvents were then stored in glass Erlenmeyer flasks under an argon atmosphere. Isoamyl bromide was distilled using short-path distillation equipment. The potassium carbonate was washed with distilled acetone prior to use. All glassware and PFA tubes were washed with distilled acetone before derivatization with isoamyl bromide. A blank test was then performed, and GC-MS analysis was carried out with increased sensitivity approximately 10 times; however, no peaks interfering with isoamyl bromide were observed (Fig. 3).

Optimization of derivatization conditions　　To optimize the derivatization conditions, we included key factors, such as the amount of isoamyl bromide, reaction temperature, and time, in the preliminary experimental design. For each treatment condition, the procedure was repeated thrice, the peak area at m/z 160 was determined, and the average values and standard deviations were calculated.

Alkyl halides, such as isoamyl bromide, are versatile compounds used in substitution and condensation reactions in organic synthesis. Tea leaves contain various components that react with alkyl halides. Therefore, we investigated the reaction conditions required to sufficiently derivatize phthalimides. First, the amount of isoamyl bromide used for derivatization was investigated. Derivatization was performed, and isoamyl bromide was added to the green tea extract, but the amount of isoamyl bromide used varied. The peak areas of the derivatives reached the optimum values when the amount of isoamyl bromide was over 0.20 mL (Fig. 4A); no obvious difference was observed when the amount of reagent used was higher than 0.20 mL (Fig. 4A). Thus, the optimum amount of isoamyl bromide for derivatization was set at 0.20 mL. We further optimized the reaction temperature for derivatization, from a room temperature of 20 °C to 80 °C. The peak area of N-isoamylphthalimide was the highest at 60 °C and 80 °C (Fig. 4B). The derivatization time was also varied from 20 to 120 min. A higher derivatization yield was achieved when the reaction time was increased from 60 to 120 min (Fig. 4C). To reduce the total analysis time, 60 min was selected as the optimal reaction time.

Fig. 3.

Mass chromatographs of N-isoamylphthalimide and the blank solution. The blank test was performed after the solvents and isoamyl bromide were distilled, potassium carbonate was washed with distilled acetone, and all glassware and perfluoro alkoxyl alkane tubes used before derivatization were washed with distilled acetone.

Fig. 4.

Effect of the amount of isoamyl bromide (A), reaction temperature (B), and reaction time (C) on the generation of N -isoamylphthalimide during tea powder extraction.

Method validation　　The proposed method for quantitative analysis was validated by determining the linearity, additive recovery rate, repeatability, reproducibility, robustness, and LOQ. Triplicate measurements of the five phthalimide standard solutions were performed for the linearity test. The linear range of the calibration curve extended from 0.10 to 1.85 μg/L when the area of the peak at m/z 160 and m/z 164 was used for quantification (r = 0.999). The linearity of these parameters was better than the following patterns: r = 0.996 for m/z 217 and m/z 221; r = 0.998 for m/z 160 and m/z 221; r = 0.998 for m/z 217 and m/z 164. Therefore, the areas of the peaks at m/z 160 and m/z 164 were selected to determine the phthalimide content. In addition, when the area of the peak at m/z 160 and m/z 164 was used for quantification, the residual value was lower than 10 %, and the P-value for the lack-of-fit test (α = 0.05) was higher than 0.05. These results confirmed the absence of curvature and linearity of the analyte responses over the tested range.

Fig. 5.

GC-MS chromatogram of N -isoamylphthalimide ( m / z 160) in the samples with the lowest level of phthalimide: 0.64 μ g/kg in green tea, 2.97 μ g/kg in oolong tea, and 2.05 μ g/kg in black tea.

Ideally, using a phthalimide-free tea sample as a blank would be desirable. However, in this study, all tea samples contained phthalimide. Therefore, the tea sample with the lowest phthalimide level for each type of tea (green, oolong, and black tea) was used for the analytical validation studies. The lowest phthalimide level in green tea was 0.64 μg/kg, that in oolong tea was 2.97 μg/kg, and that in black tea was 2.05 μg/kg. Fig. 5 shows the mass chromatogram at m/z 160 with the lowest phthalimide level in each type of tea. A phthalimide standard solution was then added to each tea sample to increase phthalimide content by 0.65, 1.30, 2.60, 10.4, or 20.8 μg/kg. Table 1 shows that the satisfactory recovery of phthalimide in all observations ranged from 81 % to 116 %, with a maximum RSD of 13 %. The data showed satisfactory repeatability and intermediate precision for each type of tea, fulfilling the requirements stipulated in the SANTE guidelines (RSD ≤ 20 %). The LOQ value was estimated based on the minimum concentration that provided adequate recovery (70–120 %) and RSD values (≤ 20 %). The LOQ for each type of tea was determined to be 0.99 μg/kg for green tea, 3.62 μg/kg for oolong tea, and 2.70 μg/kg for black tea. Robustness was defined as the mean of all recovery rates. As shown in Table 1, the robustness values of green tea, oolong tea, and black tea were 97 %, 101 %, and 98 %, respectively. The range of the phthalimide levels that met the requirements stipulated in the SANTE guidelines was 0.99 to 21.1 μg/kg for green tea, 3.62 to 23.8 μg/kg for oolong tea, and 2.70 to 23.5 μg/kg for black tea. Based on these results, the proposed method exhibited satisfactory precision and accuracy for phthalimide determination.

Phthalimide level in tea　　The proposed method was then used to analyze 337 green, 36 oolong, and 91 black tea samples (Fig. 6). All tea samples were prepared in Japan according to the corresponding packaging labels. Eight of the 337 green tea samples contained phthalimide below the LOQ (0.99 μg/kg). In addition, two oolong and four black tea samples contained phthalimide below the LOQ (3.62 μg/kg for oolong tea and 2.70 μg/kg for black tea). The maximum level of phthalimide detected was 16.2 μg/kg for green tea, 12.6 μg/kg for oolong tea, and 11.8 μg/kg for black tea. From the results of the Jarque–Bera tests with a significance level of 0.05, the frequency distribution of phthalimide in green tea, oolong tea, and black tea was not normal. Therefore, the mean phthalimide content in each tea type was not evaluated. A Mann–Whitney U test was performed, and no significant difference (p > 0.05) in phthalimide levels was found across the types of tea investigated.

Table 1. Method validation and analytical quality control based on the SANTE/11312/2021 guidelines for quantifying phthalimide levels in green tea, oolong tea, and black tea.

Type of tea	Recovery (%)		a RSD (%)		Repeatability	Intermediate precision	d LOQ	e Robustness	f Range
	min	max	min	max	b RSDrep (%)	c RSDip (%)	( μ g/kg)	(%)	( μ g/kg)
Green tea	81	115	6	12	8	9	0.99	97	0.99–21.1
Oolong tea	85	117	6	12	9	10	3.62	101	3.62–23.8
Black tea	83	116	7	13	11	11	2.70	98	2.70–23.5

^a The relative standard deviation (RSD) was obtained from five replicates in a day.

^b RSD_rep for assessing the repeatability

^c RSD_ip for assessing the intermediate precision

^d The limit of quantification (LOQ) was selected as the lowest level for which robustness was confirmed to be 70–120 % and precision RSD ≤ 20 % based on the additive recovery test.

^e Average recovery rate for the additive recovery test

^f Range that satisfied the condition for which robustness was within the additive recovery range of 70–120 %

with a precision RSD ≤ 20 % based on the additive recovery test.

Fig. 6.

Concentration of phthalimide in green tea, oolong tea, and black tea.

In Japan, folpet is registered as a pesticide for use in adzuki beans, cucumbers, tomatoes, melons, and onionsⁱ⁾. However, folpet is not listed as an applicable pesticide in tea production. The analysis of phthalimide in commercial tea samples revealed that most tea samples made in Japan contain phthalimide. Moreover, it has been reported that the runoff rate of folpet from fields is 0.02 %ⁱⁱ⁾. This suggests that most of the phthalimide in tea is not derived from folpet. Tea leaves contain significant amounts of phthalic anhydride (Wittig et al., 2022), which can easily react with ammonia to form phthalimide. Amino acids, including asparagine, aspartic acid, cysteine, and glutamine, release large amounts of ammonia at a high temperature (180 °C; Sohn and Ho, 1995). These are the amino acids in green, oolong, and black tea (Horanni and Engelhardt, 2013). Glutamine completely releases ammonia at 110 °C (Sohn and Ho, 1995). During tea manufacturing, tea leaves are dried in hot air at approximately 100 °C. Wittig et al. (2022) confirmed that phthalimide was formed by heating freeze-dried tea leaves; therefore, we assumed that phthalimide is formed during the tea manufacturing process. The range of phthalimide levels was 1.04 to 16.2 μg/kg for tea leaves made in Japan (Fig. 6). The phthalimide level in tea leaves varies widely. However, little is known about how phthalimide forms in the tea manufacturing process, such as during steaming and pan-frying. In future studies, we plan to clarify the formation of phthalimide during the tea manufacturing process and its relationship with phthalimide precursors, phthalic acid, and phthalic anhydride.

Conclusions

A new analytical method was developed to determine the phthalimide content in tea. This method was based on acetone extraction and derivatization using isoamyl bromide coupled with GC-MS. The proposed method showed satisfactory precision and accuracy for determining the phthalimide content in tea. These observations provide crucial information and an analytical method to further study the sources and related pathways of phthalimide formation in tea.

Acknowledgements　　This research was financially supported by agricultural, forestry, and fishery products and food export promotion organization grants from the Japan Tea Central Public Interest Incorporated Association.

Conflict of interest　　There are no conflicts of interest to declare.

Abbreviations

GC

Gas chromatography

MS

Mass spectrometry

LC

Liquid chromatography

PFA

Perfluoroalkoxyl alkane

SPE

Solid phase extraction

i.d.

internal diameter

BPTAB

3-bromopropyltrimethylammonium bromide

LOQ

limit of quantification

RSD

relative standard deviation

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