Rapid Quantitative Determination of Isoprene Monomer in Living Taraxacum kok-sag

Xiang Tong; Guo Tianyang; Zhang Xi; Chen Yunhan;Dong Yiyang; Zhang Jichuan; Ma Qiang; Zhang Liqun

(1. Center of Adνanced Elastomer Materials, College of Materials and Engineering,Beijing Uniνersity of Chemical Technology, Beijing 100029;2. College of Life Science and Technology, Beijing Uniνersity of Chemical Technology, Beijing 100029;3. School of Food and Health, Beijing Technology and Business Uniνersity, Beijing 100048;4. Institute of Industrial and Consumer Product Safety, Chinese Academy of Inspection and Quarantine, Beijing 100176)

Abstract: Taraxacum kok-saghyz (TKS) is rich in natural rubber (NR), a natural organic macromolecular compound composed of cis-1,4-polyisoprene, and may become the second NR-bearing plant for biochemical engineering development.In this paper, a rapid and quantitative ultra-high performance liquid chromatography tandem mass spectrometry (UHPLCMS/MS) method was established for determination of macromolecular biosynthesis substrate (dimethylallyl pyrophosphate,DMAPP) and initiator (farnesyl pyrophosphate, FPP) contained in TKS. A Kromasil C18 chromatographic column was used for separation, and the multi-reaction monitoring mode (MRM) of triple quadrupole mass spectrometry was used for detection. Quanti fication was performed by external calibration method. The results showed that the limit of detection (LOD)and the limit of quantitation (LOQ) of DMAPP were 2.42 μg/L and 7.26 μg/L, respectively, and the LOQ and the LOD of FPP were 1.02 μg/L and 3.05 μg/L, respectively. At a concentration of 1—1000 μg/L, both analytes had good determination coefficients (＞ 0.999) of calibration curve. The recoveries of DMAPP and FPP were between 99.0% and 117.1%. In real samples detection, the contents of DMAPP and FPP in TKS samples were between 23.32—82.77 μg/L and 12.03—85.67 μg/L, respectively. Thus, this approach is a reliable method to quantify DMAPP and FPP in TKS.

Key words: quantitation; Taraxacum kok-saghyz (TKS); isoprenoids; ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS); natural rubber (NR)

1 Introduction

Natural rubber (NR) has the unique physical and chemical properties such as high elasticity, good wear resistance,and strain-induced crystallization[1], which cannot be superseded by synthetic rubber. NR has been widely used in more than 50000 products, including aerospace, military,medical materials, building materials, and automotive industry[2]. Although more than 2000 known plants contain NR, only Brazilian Hevea rubber has been successfully commercialized at present[3]. Coupled with factors such as leaf blight in South America, the future supply pressure of NR will increase sharply. In order to relieve the pressure,the trend of developing second NR is more urgent[4-5].

Taraxacum kok-saghyz(TKS), also known as Rubber grass, Rubber Dandelion or Russian Dandelion, is an annual or perennial herbaceous plant, whose root contains 6%—28% of dry NR mass, which is suitable for largescale mechanized planting that can be less affected by territory, and has higher resistance to diseases and insect pests[6]. Nowadays, TKS has been cultivated in Xinjiang,Gansu, Heilongjiang, and Hainan in China[7].

It has been proposed that NR in Hevea can be synthesized in vitro, so it is particularly important to study the biosynthesis process of NR[8]. The research on NR biosynthesis started in the 1950s and a great progress has been made so far. Rubber molecules are the metabolic end products of isoprenoids in rubber-producing plants[9].At present, more than 29000 kinds of isoprenoids have been identified, some of which are necessary for plant development, e.g., gibberellin, abscisic acid[10], and some of which are produced by interactions between plants and environment, e.g., phytoalexins; however,the physiological functions in plants are not clear[11].Although the biochemical reaction steps and enzymes related to the reaction in the early and middle stages of rubber macromolecule synthesis have been basically understood, little is known about the detailed formation process of rubber macromolecules.

NR is a kind of linear biomacromolecules produced by polymerization of isopentenyl pyrophosphate (IPP)monomers. In higher plants, IPP molecules are synthesized mainly through the mevalonate pathway (MVA), and are then isomerized to its isomer — dimethylallyl pyrophosphate (DMAPP) under the action of isopentenyl transferase (IPT)[12]. With IPP and DMAPP serving as monomers, a series of isoprenoids homologues such as geranyl pyrophosphate (GPP), farnesyl pyrophosphate(FPP) and geranylgeranyl pyrophosphate (GGPP)can be formed under the combined action of rubber transferase (RuT) and other enzymes. Finally, a linear rubber macromolecule was formed under the action of various enzymes[13]. Studies have shown that isoprenoid homologues in the biosynthesis of rubber macromolecules can be used as initiators to synthesize rubber macromolecules, while FPP is a representative[14].In vitro studies have shown that the biosynthesis rate and the molecular weight of rubber macromolecules depend on the substrate concentration, especially the concentration of DMAPP. In living plants, the concentration level of isoprenoids is very low, and it also has high biological activity. It is very difficult to monitor the content of such substances[15]. Generally, isoprenoids substances are usually proliferated by cell culture, labeled by isotopes or extracted after derivatization[16], and finally analyzed by fluorescence,chromatography, mass spectrometry, etc.[17]Some of these methods are costly and cumbersome, coupled with low sensitivity and poor reliability[18].

In this paper, the isoprenoids in the roots of TKS were determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLCQqQ MS). The simple external calibration method can be used for the direct and real-time qualitative and quantitative detection of isoprenoids substances in TKS. It can also provide the data support for TKS seed selection.

2 Experimental

2.1 Materials

Dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP) having a purity of 99.9% were purchased from Sigma-Aldrich (Munich, Germany).The HPLC-grade methanol, acetonitrile, and ammonium hydroxide were purchased from the Beijing Chemical Industry Group Co., Ltd. (Beijing, China). Ultrapure water was obtained by using a Milli-Q UP Water Purification System (MA, USA). Nitrogen was obtained by a XTNS-1 full-automatic nitrogen blowing concentrator. TKS plants were cultivated in Xinjiang Autonomous Region, Hainan province, and Heilongjiang province, and were moved to Beijing before investigation.

Standard stock solution: Two types of isoprenoids standards were prepared into standard stock solutions of 500 mg/L with HPLC-grade methanol for use.

2.2 Instrument condition

DMAPP and FPP were detected by the Waters ACQUIT ultra-high performance liquid chromatography (Waters Corporation, Milford, MA, USA) integrated with a Xevo TQ-MS triple quadrupole mass spectrometer (Manchester,UK). The obtained data were processed by a MassLynx4.1 data processing system.

The Ultra-high performance liquid chromatography was equipped with achromatographic column, Kromasil C18column (4.6 mm×50 mm×3.5 μm); operating at a temperature of 30 °C. The mobile phase: A was a 0.1%(by volume) ammonium hydroxide solution; B was acetonitrile. Gradient elution conditions were as follows:0—4.5 min, 98% A to 95% A; 4.5—7 min, 95% A to 0%A; 7—8 min, 0% A to 50% A; 8—9 min, 50% A to 98% A;9—10 min, equilibration with 98% A. The flow rate was 0.3 mL/min and the injection volume was 2 μL.

Mass spectrometry conditions were as follows. Ion source: electrospray ion source (ESI); ion mode: negative;capillary voltage: 2.5 kV; ion source temperature:150 °C; desolvation gas type: nitrogen, desolvation gas temperature: 500 °C, desolvation gas flow rate: 1 000 L/h;collision gas: argon; collision gas flow rate: 0.14 mL/min;data acquisition mode: multiple reaction monitoring(MRM).

2.3 Calibration curves

DMAPP and FPP standard reserve solutions (500 mg/L)were diluted with corresponding solvents methanol/acetonitrile/water (with a volume ratio of 1:1:1) into different calibration solutions, at gradient concentrations of 0.01, 0.05, 0.1, 0.2, 0.5 and 1 mg/L, respectively. The optimized method was used to test, and the calibration curve was obtained to determine the concentration of each compound in TKS sample.

2.4 Sample preparation

The living plants of TKS have abundant bioactive substances. They were provided by the cultivation units and planted in the soil flowerpot before the experiments.The plant was dug out and the soil in root was washed away with tap water. Then the root was rinsed with the ultra-pure water and air dried. Three different extraction solvents were compared: methanol, acetonitrile, and 0.1%ammonium hydroxide. The specific extraction method was as follows: The root samples of TKS were cut to ca.3 cm long (approximately 1 g) and soaked in 10 mL of extractive solvent for 3—5 min. Then the mixture was shaken by a vortex oscillator three times for 2 min each time prior to being ultrasonically extracted under 400 W for 20 min, and centrifuged with a rotational velocity of 12 000 r/min for 20 minutes at 4 °C. Each group of samples were repeated more than three times to ensure the complete extraction of the target product, and then 30 mL of the supernatant was concentrated to 15 mL by nitrogen blowing under a pressure of 0.5 MPa in water bath at 30 °C, and finally was filtered with a 0.22 μm membrane before injection[19].

3 Results and Discussion

3.1 Optimization of LC-MS conditions

3.1.1 Selection of target substances

The biosynthesis of NR macromolecules is a complex biochemical process starting from DMAPP or IPP, and isoprenoid homologues can initiate and participate in the reaction. It is generally believed that FPP has the highest initiation efficiency. The content of various isoprenoids in plants is closely related to the rate of biosynthesis and the molecular weight of the rubber molecules eventually formed[20]. In this experiment, DMAPP and FPP were selected as the detection targets, and the content of DMAPP and FPP in the test substance can also be used as the referral content values of other isoprenoid homologues.

3.1.2 Optimization of liquid chromatography and mass spectrometry conditions

The mixed standard storage solution was diluted to 1 mg/L for the optimization of liquid chromatography and mass spectrometry conditions. Mobile phase A: Ultra-pure water, acidic ammonium acetate, alkaline ammonium bicarbonate, 0.1% formic acid water (volume ratio) and 0.1% ammonia hydroxide (volume ratio) were tested as mobile phase separately. Finally, the 0.1% ammonia water was chosen as the mobile phase according to chromatographic peak shape and responsive intensity.

The general injection volume of the instrument used was 2 μL, and the maximum injection volume was 7.5 μL under the existing loop condition. An injection volume of 1 μL, 2 μL, 5 μL, and 7.5 μL, respectively, was compared and analyzed by the half-width and the response intensity.Finally, the common 2 μL injection volume was chosen.

A column temperature of 30 °C, 40 °C, and 50 °C,respectively, was tested, which showed the similar results.Upon considering the actual conditions of samples tested,a temperature of 30 °C was considered as the optimum column temperature eventually[21]. The peaks of DMAPP and FPP identified by UHPLC-QqQ MS under multiple reaction monitoring (MRM) mode are shown in Figure 1.The intelligent auto function in MassLynx4.1 was used to determine the standard mass spectrum parameters, and the mass spectrum parameters of the two substances obtained thereby are shown in Table 1.

Figure 1 The peaks of dimethylallyl pyrophosphate(DMAPP) and farnesyl pyrophosphate (FPP) at the 1 000 μg/L level detected by UHPLC-QqQ MS under multiple reaction monitoring (MRM) mode

Table 1 MS parameters of dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP) identi fied by UHPLC-QqQ MS

3.2 Calibration curves, LODs and LOQs

Under the working conditions of the instrument, 6—7 different concentrations of mixed standard solutions of the two compounds were set for the analysis, and the total ion chromatography (TIC) at different concentrations were obtained. Then the ion pairs were extracted to determine the quantitative ion pairs and were used to calculate their peak areas, while each concentration of the sample was measured in triplicate. The calibration curve was obtained by taking the mass concentration of the compound as abscissa and the peak area as ordinate.The calibration curve and linear regression parameters are shown in Figure 2. The determination coefficients of the two standards areR2=0.9991 (for DMAPP) andR2=0.9995 (for FPP). By measuring the solvent system without standard addition, the noise of the signal was obtained. The limits of detection (LODs) and the limits of quantitation (LOQs) of two analytes were determined by 3 times the signal to noise ratio and 10 times the signal to noise ratio, respectively[23], as shown in Table 2.

Figure 2 Calibration curves of dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP) by UHPLC-QqQ MS

Table 2 LOD and LOQ of dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP) measured by UHPLC-QqQ MS

3.3 Measurement investigation

The experimental sample is a complete plant of TKS,which is a complex biological system. Most of TKS exists in the root of plant with a relatively low concentration,which makes it difficult to extract and enrich the target substances to be measured. According to the properties of the substances to be measured, methanol was used as the extracting solvent, while acetonitrile was used as the deproteinizer. Upon considering the biological water environment of the plant itself, a certain amount of water was added into the extracting solvent to promote the extraction. Different extraction solvents including methanol, acetonitrile, methanol/acetonitrile(at a volume ratio of 1:1), methanol/acetonitrile/water/ammonium hydroxide (at a volume ratio of 1:1:1:0.001)were compared, which showed that methanol was better for extraction of DMAPP, while acetonitrile was better for extraction of FPP. In view of the water environment,which needs a weak alkalinity of the organism itself coupled with the use of acetonitrile as the protein remover, the final extraction solvent was a mixture of methanol/acetonitrile/water/ammonium hydroxide (at a volume ratio of 1:1:1:0.001), During the experiment,the same sample was extracted three times with the extraction solvent. It was found that the content of the substance to be measured in the methanol/acetonitrile/water/ammonium hydroxide (at a volume ratio of 1:1:1:0.001) was below LOD, and then it was considered that the extraction was completed. Because the content of the substance to be tested was extremely low in the TKS, the extraction solution was subjected to nitrogen blowing to increase the concentration of the analyte. The optimized LC-MS/MS method was used for the detection.Each group of samples was injected in triplicate, and the average value was taken into the calibration curve to obtain the corresponding analyte content, as illustrated in Figure 3 and Table 3.

The results showed the DMAPP and FPP contents were lower in TKS and had a great individual variance. The DMAPP content was greater than or close to the FPP content.

Daytime Repeatability Test: the same sample was stored for 48 hours before the same test. Similarly, 3 parallel tests were taken to calculate the average value of products. The results in Table 4 showed that the content of DMAPP and FPP was signi ficantly reduced. The FPP content in samples No. 4 and No. 5 was lower than LOQ,but the reduction of DMAPP was less than FPP, indicating that the stability of DMAPP was better than FPP. The outcome of analyses may be ascribed to the fact that FPP was the initiator, the activity of which was greater than that of the DMAPP monomer.

Figure 3 Total ion chromatography (TIC) and ion extraction chromatography (EIC) of dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP)in the sample TKS-1 measured by UHPLC-QqQ MS

Table 3 Detection of dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP) in TKSs by UHPLC-QqQ MS

3.4 Recovery and precision experiment

TKS without NR was used as a blank sample for the spiked recovery experiment. The three spiked levels of low, medium, and high values were set. The average value was obtained in tuplicate for each level. The recoveries of DMAPP and FPP were calculated[23]as shown in Table 5.The test results showed that the recovery of this method was between 99%—117.1%, which demonstrated a good analytical accuracy.

4 Conclusions

A rapid, accurate, and efficient method for the quantitative determination of natural rubber macromolecular biosynthesis precursors in TKS was established. Two important precursors in natural rubber biosynthesis,dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP), were detected. The results showed that the limits of detection (LODs) of DMAPP and FPP were 2.42 μg/L and 1.02 μg/L, respectively, and the limits of quantitation (LOQs) of FPP were 7.26 μg/L and 3.05 μg/L, respecively. The recovery of two analytes were between 99.0% and 117.1%. The daytime repeatability test had found that the stability of DMAPP as the main monomer was greater than that of the main initiator FPP.The concentration ranges for DMAPP and FPP were 23.32—82.77 μg/L and 12.03—85.67 μg/L, respectively.Finally, this method has provided a theoretical support for the biosynthesis mechanism of NR to lay a foundation for the final biosynthesis of NR in vitro.

Table 4 Concentration of dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP) in TKSs measured by UHPLC-QqQ MS after being stored for 48 hours since extraction of samples

Table 5 Results of tests on precision and recovery of dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP) (n=6)

Acknowledgement:The authors acknowledge the supports of the National Key Research and Development of Bio-Based Rubber (2017YFB0306900 & 2017YFB0306901), the National Natural Science Foundation of China (51673012),the Fundamental Research Funds for the Central Universities(PYBZ1828), the Beijing Technology and Business Universtiy Youth Scholoars Funds (PXM2019_014213_000007).