Direct Determination of 2-Acetyl-1-Pyrroline in Rice by Ultrasound-Assisted Solvent Extraction Coupled with Ultra-performance Liquid Chromatography-Tandem Mass Spectrometry

In this paper, we present a novel direct detection method for the characteristic fragrant component 2-acetyl-1-pyrroline (2-AP) in rice by using ultrasound-assisted solvent extraction (UASE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). No tedious derivatization procedures and toxic solvents were involved in the sample preparation process. 2-AP was extracted with ethanol by UASE from rice samples, and remained good stability within 27 h. By direct injection to UPLC-MS/MS, accurate results were obtained through matrix-matched internal standard calibration. Under optimized conditions, good repeatability (2.8%), reproducibility (7.6%), and recoveries (83.9–108%) were achieved with the limit of detection down to 0.3 μg/kg. The results of 2-AP in 13 rice samples determined by the present method were in the range of 19.4–124.0 μg/kg. The sensitivity and reliability of the developed UASE UPLC-MS/MS method are comparable with previously published SPME GC–MS/MS methods or derivatization-based HPLC–MS/MS method. High analysis throughput, low laboriousness, excellent accuracies, and low toxic solvent consumption are the main characteristics of the newly presented method.


Introduction
2-Acetyl-1-pyrroline (2-AP), with the International Union of Pure and Applied Chemistry (IUPAC) name 1-(3, 4-dihydro-2H-pyrrol-5-yl) ethanone, is considered to be one of the major contributors to the unique fragrance of scented rice since it was isolated and identified in aromatic rice in 1982 (Buttery et al. 1982). The content of 2-AP in rice determines its value not only in the global market but also in the consumer demand and the farming practices (Verma and Srivastav 2022). To investigate the concentration levels, the synthetic mechanisms, and the relevant influencing parameters in aromatic or non-aromatic rice, many researchers emphasized on how to accurately detect and confirm this particular compound. According to the published literature, the concentrations are usually as low as sub ppb (μg/kg) in aromatic rice and even tenfold lower in non-aromatic rice (Routray and Rayaguru 2017;Wakte et al. 2017;Wei et al. 2017). Therefore, sample treatment procedures including extraction, purification, and concentration are essential to obtain reliable assay results. The procedures consist of conventional and modern methods. Conventional methods mainly employ direct distillation, steam distillation-solvent extraction, and direct solvent extraction, while modern methods frequently use headspace-solid phase micro-extraction (HS SPME), static headspace, and stir bar sorptive extraction (Twister) (Wakte et al. 2017). Generally, these extraction methods are coupled to gas chromatograph (GC) with FID, NPD, MS, TOF MS, or tandem MS detectors (Bergman et al. 2000;Bryant and McClung 2011;Grimm et al. 2001Grimm et al. , 2011Guo et al. 2020;Hopfer et al. 2016;Lee et al. 2019;Maraval et al. 2010;Mathure et al. 2011;Sriseadka et al. 2006;Wei et al. 2021). Until recently, Jost et al. proposed a new method based on derivatization and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the determination of 2-AP in foods (including rice) (Jost et al. 2019), and Bösl et al. also developed a method based on derivatization by 3-nitrophenyhydrazine coupled to LC-MS/MS analysis to quantitation of odor-active 2-acetyl azaheterocycles in food products including cooked jasmine rice in 2021 (Bösl et al. 2021).
Many factors might affect the quantitation accuracy during both the sample preparation and instrument analysis because of the problems such as the instability and unavoidable loss of 2-AP (Hausch et al. 2018;Wei et al. 2017), the co-elution of 2-AP with similar compounds (Sriseadka et al. 2006), the impossibility of achieving equilibrium between the internal standard and sample matrix (Cadwallader et al. 2016), the new generation of 2-AP at high temperatures (Hopfer et al. 2016), and so on. Additionally, the labor intensive and time-consuming procedures or one by one SPME operation lower the analysis throughput and limit the application of these techniques. Therefore, developing new methods for the determination of 2-AP and other related researches targeting on the above problems and troubleshootings are still very active.
Ultrasound-assisted solvent extraction (UASE) is an efficient technique for enhancing extraction of components from plant and animal materials (Vilkhu et al. 2008), the inexpensiveness and simple operation makes it a good alternative to some traditional extraction methods. The mechanism of UASE is attributed to mechanical and cavitation effects which can result in particle size reduction, and enhanced mass transfer (Khoei and Chekin 2016). Ethanol is frequently employed as the extraction solvent due to its low cost and environmentally benign characteristic. Furthermore, as far as the extraction yield of 2-AP is concerned, much higher yield can be achieved by using ethanol as extraction solvent directly than using supercritical carbon dioxide extraction and simultaneous steam distillation extraction from P. amaryllifolius leaves according to Laohakunjit and Noomhorm's research (Laohakunjit and Noomhorm 2004), and a similar result was also obtained by Azhar et al. when they extracted 2-AP from Pandanus amaryllifolius Roxb. by using ethanol as solvent in last year (Azhar et al. 2022). Additionally, according to the results reported by Rungsardthong and Noomhoom, 2-AP kept good stability with 92% remained in ethanol in 14 days (Rungsardthong and Noomhoom 2005).
In this study, we aim to present an UASE and nonderivatization technique coupled with UPLC-MS/MS analysis for the accurate determination of 2-AP in rice. By optimizing the pertinent parameters affecting the extraction efficiencies and the determination performance, appropriate conditions were set up. The results of 13 rice samples obtained by the present method are generally consistent with those reported in published journals. In particular, the limit of detection, the repeatability, and the recoveries are comparable with those of previously published methods by using the proposed UASE UPLC-MS/MS method. Furthermore, the high analysis throughput and non-derivatization step enable this method to be applied in batch analysis of 2-AP in massive rice samples.

Rice Samples
The 13 rice samples were all purchased from local supermarkets in Xi'an, China. All of them were claimed as the "fragrant rice" on the packages and stored at 4 °C with the packages remained intact prior to analysis. Additionally, a regular non-fragrant rice which did not contain 2-AP (less than the LOD) by pre-verification was purchased from a local supermarket in Xi'an as well, and this regular rice was used to make the matrix-matched solutions throughout the research.

Preparation of Calibration Standard Solutions and Calibration Curves
2-AP stock standard solution (1000 μg/mL) was made by dissolving 1 mg of 2-AP in 1 mL of ethanol and stored at − 18 °C. The 2-AP working solutions (1 μg/mL, 10 μg/ mL) were progressively diluted with ethanol by using the stock standard solution prior to use. The 10 mg of TMP was weighed and dissolved in 10 mL of ethanol to make the TMP stock internal standard solution (1000 μg/mL) and also stored at − 18 °C. The regular non-fragrant rice was employed as the matrix blank to prepare the matrix-matched calibration standard solutions. The standard solutions at various concentrations (0, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000 ng/mL, equivalent to 0, 0.3, 0.75, 1.5, 3.0, 7.5, 15, 30, 75, 150, 300 µg/kg in matrix-matched standards) were prepared by pipetting corresponding amount of the 2-AP working solutions (or further diluted solutions) as required to the rice samples, and each calibration standard solution contained 400 ng/mL (equivalent to 120 µg/kg in rice samples) of TMP. Then these matrix-matched standards were extracted and pretreated under the subsequent optimized conditions. The calibration curve for UPLC-MS/MS analysis was made by plotting the ratios of peak areas of 2-AP to those of TMP versus the corresponding concentrations of 2-AP.

UPLC-MS/MS Analysis
Separation and quantification of 2-AP and TMP were performed by using an ultra-performance liquid chromatography (ExionLC, SCIEX, USA), equipped with a binary gradient AC pump and an automatic injector with temperature set at 4 °C, coupled to a linear ion trap triple-stage quadrupole mass spectrometer (API 5500 QTrap, SCIEX, USA). The instrument control and data processing were conducted with Analyst software (Ver. 1.6.3, SCIEX, USA). The injection volume was set at 5.0 μL. The chromatographic separation was carried out with a Waters Cortecs T3 column (2.7 μm, 100 mm × 2.1 mm) (Waters, Ireland) at a flow rate of 0.3 mL/min. The column temperature was kept at 40 °C. The mobile phases were (A) 0.1% (v/v) formic acid in ultrapure water and (B) 0.1% (v/v) formic acid in acetonitrile with the following gradient: from 20 to 80% (B) in 4 min starting at 1.0 min; keeping 80% (B) until 5.5 min; from 80 to 20% (B) in 0.1 min; keeping 20% (B) for 4.4 min.
Mass analysis was performed with an ESI source in the positive (ESI +) ion mode. Nitrogen was used as the nebulizer gas. The optimum MS parameters were curtain gas, 35 psi; collision gas, 2 psi; ion source gas 1, 55 psi; ion source gas 2, 55 psi; ion spray voltage, 5500 V; and turbo spray ion source temperature, 550 °C. All analyses were analyzed in multiple reaction monitoring (MRM) mode. The UPLC-MS/MS settings and parameters of ESI source were optimized by manual infusion of standards with a syringe pump. The optimized parameters are summarized as follows: (1)  (2) for TMP, the precursor of [TMP + H] + generated Q1 (m/z: 121.6), and production ions of Q3 (m/z: 106.1 and 79.2) obtained by CE set at 39 V and 32 V, respectively, and the transition of 121.6/106.1 was selected for quantitation. Other parameters for 2-AP and TMP were same: declustering potentials (DP), 80 V; entrance potentials (EP), 10 V; collision cell exit potentials (CXP), 11 V; and the dwell times for all the transitions were 100 ms. For quantitation, 2-AP concentrations in the samples were calculated from the peak-area ratios between 2-AP (112.0/70.0) and TMP (121.6/106.1) based on the internal standard calibration curve. In addition, the relative intensities (% of base peak) were used to help the identification and confirmation of 2-AP according to the maximum permitted tolerance values in 2002/657/EC. In this study, the relative intensities of 2-AP (112.0/43.0) were about 22.4% at the content of 20 µg/kg. Therefore, the permitted tolerances are ± 25% of the 22.4%.

UASE and Sample Treatment
Ethanol was selected as the extraction solvent in this study because it can keep good stability and high extraction efficiency for 2-AP. The related parameters that might affect extraction efficiencies such as ultrasonic frequencies (45 Hz, 80 Hz, and 100 Hz), extraction time (20 min, 30 min, 40 min, 50 min, and 60 min), extraction temperature (20 °C, 30 °C, 40 °C, 50 °C, 60 °C), the ratio of samples to the solvent (1:2, 1:3, 1:4, 1:5, 1:6, m/v), and cycles of repetitive extraction (1 to 5) were investigated and selected with univariate analysis through extraction of one specific rice sample containing about 100 μg/kg of 2-AP, and all of the extraction experiments were carried out in triplicate. After the extraction procedures were completed, the samples were further pretreated with nitrogen blowing concentrator and syringe filter, then submitted to the UPLC-MS/MS analysis. The peak areas of the quantitation transition were employed to determine the optimized extraction conditions. The optimal extraction conditions were described as follows: ultrasonic frequency, 45 kHz (input power: 500 w); extraction time, 60 min; extraction temperature, 60 °C; and the ratio of sample to solvent in total, 1:5 (w/v). Because the repetitive extraction cycles have crucial effects on the maximization of the target compound, various cycles (1 to 5) were investigated. The different extraction cycles meant the extraction solvent was divided into different portions to carry out the extraction. In the experiments, 25 mL of solvent was used for one cycle; similarly, 12.5 mL plus 12.5 mL for 2 cycles, 10 mL plus 7.5 mL and 7.5 mL for 3 cycles, 10 mL plus 3 times of 5 mL for 4 cycles, and 5 times of 5 mL for 5 cycles. Figure 1 shows that 3 to 5 extraction cycles can provide satisfied efficiencies. Considering the time cost and solvent consumption, 3 cycles were selected for the subsequent experiments. That means the whole extraction time was 180 min, this result is well consistent with the research conducted by Cadwallader et al., their recommended extraction time was 120-150 min when using solvent extraction (Cadwallader et al. 2016).
The detailed sample preparation process was described as follows: rice samples were ground with an electric grinder at low or medium speed to minimize the temperature increase and the loss of 2-AP during grinding. The samples were then passed through a mesh sieve. Then, 5.0 g of above ground rice sample was weighed into a 50 mL conical flask, and 600 ng of TMP was added in the flask, after 10 mL of ethanol was added, the flask was capped and sealed with parafilm and placed in the ultrasonic water bath for extraction by setting the temperature at 60 °C which was controlled through a circulating water supply system with a thermostat at the temperature. When the first-cycle extraction was completed, the flask was taken out and stood for settlement, and the supernatant was transferred to a 25 mL centrifuge tube. The extraction was repeated twice by adding 7.5 mL of ethanol each time. The supernatants obtained by 3 extraction cycles were merged and centrifuged for 20 min at 15,000 rpm (RFC: 21,130 g) with the centrifuge temperature setting at 10 °C. The resultant supernatant was transferred to a new centrifuge tube and evaporated with a nitrogen blowing concentrator to the volume less than 1.5 mL, and the final volume was set to 1.5 mL with ethanol, and then filtered into an LC vial by passing through a 0.22 μm PTFE syringe filter. The prepared samples were subjected to UPLC-MS/MS analysis. For the recovery experiments, after the samples were weighed and TMP was added, 50, 100, and 200 ng of 2-AP were also added into three samples, respectively, then other procedures were same as the above rice sample extraction. All of sample preparation and determination were in triplicate unless otherwise specified.

Effects of Temperature and Precursors on the Formation of 2-AP
Temperature plays an important role during the solvent extraction; generally, higher temperature can accelerate the mass transfer and shorten the equilibration time. However, for the extraction of 2-AP, the new formation cannot be avoided at elevated temperature, especially when the temperature is over 80 °C, Maillard reactions between proline (as the precursor of 2-AP synthesis) and sugar degradation products may result in the production of 2-AP (Hopfer et al. 2016;Yoshihashi et al. 2002). Thus, we reinvestigated the effects of extraction temperature and 2-AP precursors. With the temperature remained at 60 °C, 0.2-2% (w/w) of L-proline or L-glutamic acid were added into the 5.0 g of rice samples. After extraction under the optimal conditions, the samples were subjected Fig. 1 Effect of extraction cycles for the ultrasound-assisted solvent extraction. Ultrasonic frequency, 45 kHz (input power: 500 w); extraction time, 60 min; extraction temperature, 60 °C; the ratio of sample to solvent, 1:5 (w/v). The numbers on the columns are the statistical mean differences (n = 3) to UPLC-MS/MS analysis, and the responses of 2-AP were compared. The results revealed that no significant increases were found even 2% (w/w) of L-proline or L-glutamic acid was added. This can be ascribed to the organic solvent circumstance wherein ethanol denatured the rice proteins and prevented the formation of 2-AP at 60 °C.

Stability of 2-AP
The stability of 2-AP is one of most important factors that influence the accuracy of determination especially for the analysis of aqueous solutions and sample matrix through headspace or steam distillation techniques. To investigate the stability of 2-AP in the rice sample matrix, 50 ng/mL of 2-AP prepared with the rice sample blank matrix (equivalent to 15 μg/kg in rice) was discretely injected to the UPLC-MS/ MS in every 2 or 3 h during the batch sample analysis. The results revealed that more than 95% of original 2-AP could be detected within 27 h, which is consistent with the research by Rungsardthong and Noomhoom (2005). Additionally, by comparisons of the ratios of 2-AP peak areas to TMP peak areas that 2-AP standard dissolved in ethanol and in the matrix-matched solution, we found the ratios almost kept at a relatively stable value. During the ultrasonic extraction and nitrogen blowing concentration, even though the degradation and loss of volatile 2-AP were inevitable, the matrixmatched standards and the internal standard of TMP could make compensations for the loss of 2-AP. Furthermore, 2-AP and TMP have similar boiling point (about 170 °C at 760 mmHg), but the boiling point of ethanol as the solvent is about 83 °C (at 760 mmHg). Therefore, most of 2-AP and TMP can be retained in the concentrated solution during the nitrogen-blowing concentration process due to the big differences of boiling points between the solvent and the solutes. Table 1 summarizes the characteristics of the proposed methods including the precisions, recoveries, linearity, limit of detection (LOD), and limit of quantification (LOQ). The precisions of intra-assay were obtained by detecting the rice samples which contained about 20 µg/kg in 5 replications in 1 day, and the precisions of inter-assay were obtained in 5 days through detecting the above same rice samples. The recoveries were investigated by spiked 3 concentration levels of 2-AP standard (high, medium, and low, where medium is close to the content in the sample, high is double of the content, and low is half of the content). The linearity range and coefficient of determination were determined by making the calibration curve with least square method. The LOD and LOQ were determined based on the ratio of signal to noise (3 and 10, respectively). The relatively big deviation of inter-assay might be caused by the instability of 2-AP in the rice once the packages were opened even though they were stored at low temperature.

The Performance of the Proposed Methods and Real Sample Analysis
To further demonstrate the comprehensive performance of the newly developed method, detailed comparisons of the present UPLC-MS/MS method with the previously published methods for 2-AP determination are shown in Table 2. The UASE UPLC-MS/MS method provides a comparative sensitivity in comparison with the HS SPME GC-MS/MS methods and the OPD derivatization-based HPLC-MS/MS method. Due to the benefits of batch process for multi-sample treatment and analysis, high analysis throughput could be achieved compared with single-sample SPME coupled with GC-MS/MS analysis. In addition, no any derivatization procedures were involved in the sample preparation.
To validate the applicability of the developed method to real sample analysis, 13 fragrant rice samples were analyzed with the developed UASE UPLC-MS/MS method, and each sample was determined in triplicate. Among the 13 rice samples, although they are different varieties such as long grain, medium grain, or different ecotypes like indica or japonica, the contents of starch are similar (approximately 90% in milled rice grain, Bonto et al. 2021) even the amylose and amylopectin might be different. After 180 min of ultrasonic extraction with ethanol, the natural structure might be destroyed by the strong acoustic cavitation, and 2-AP can be released into the solvent as much as possible due to its high dissolubility and stability in the polar solvent such as ethanol and methanol. For further investigation of the extraction yield of 2-AP from different types of rice, one long grain and one medium grain were selected to compare the recoveries, the results indicate the similar recoveries (90.1% and 91.5%, respectively) were obtained when the spiked contents were at the levels that the samples contained 2-AP. The results of the 13 fragrant rice samples are summarized in Table 3. And the typical chromatograms are shown in Fig. 2. According to the presented data in the tables, good separations shown on the chromatograms, we can see it only takes a few hours for the analysis of a batch of rice samples (including extraction, nitrogen-blowing concentration, and UPLC-MS/MS analysis), the tedious derivatization procedures which need 20 h, and dark circumstances can be simplified. But still provided the comparable limit of detection and quantitation. Consequently, we can conclude that the present method, in particular, is suitable for the determination of 2-AP in rice samples, especially for the batch analysis of massive samples.

Conclusions
The presented results indicate that the developed methods based on ultrasound-assisted solvent extraction and non-derivatization technique coupled with UPLC-MS/MS detection are reliable  and sensitive for the determination of 2-acetyl-1-pyrroline in rice samples. In particular, to the best of our knowledge, the UASE UPLC-MS/MS method was the first non-derivatization-based LC-MS/MS method to carry out the determination of 2-AP in rice. The high sample analysis throughput, sufficient extraction cycles, and extraction time make these newly developed methods have more advantages such as low laboriousness, excellent accuracies, and easy operation. As the first direct detection method, in the future, it will facilitate the research on the formation mechanism of 2-AP during Maillard reaction or biosynthesis through monitoring changes of the characteristic compound, 2-acetyl-1-pyrroline. Furthermore, it will be applied to grade the fragrant rice quality in international trade when the odor is taken as a key index into consideration.