Optimization Of Extraction Solvents

Published Date: 02 Nov 2017

Introduction

Globalization of the commerce in the agricultural and livestock products has contributed to the awareness about potential threat involved and has increased the research of myotoxins. T-2 toxin, HT-2 toxin and diacetoxyscirpenol (DAS) are toxic secondary metabolites produced by Fusarium that threat seriously human and animal health all around the world. All of the three toxins belong to trchothecenes, and they are a group of structurally related mycotoxins which can be classified into four types according to the presence or absence of characteristic functional groups. T-2 toxin, HT-2 toxin and diacetoxyscirpenol (DAS) belong to Type A which are all high toxic mycotoxins in the trchothecenes family[1]. For animals Type A have been evaluated as generally toxic, immunotoxic and cytotoxic and they can inhibit protein synthesis as well as DNA synthesis both in vitro and in vivo[2-4]. The symptoms of tricothecene type A toxins (T-2, HT-2 and DAS) have been described as causing oral lesions and reduced growth and ingestation in chicken[4,5].

The three toxins can be detected from the crops contaminated by Fusarium that infecting them during growth, during and subsequent storage[6]. Fusarium poae has reported as natural contaminant mainly on grains and producer of Type A group such as T-2 toxin, HT-2 toxn and DAS[7,8]. After ingestation of contaminated crops, the broiler can be caused severe toxic efficiency by the mycotoxins. Moreover residue of toxins may be distributed in the different tissues all over the organism. It can threat human health if these toxic tissues were used as the animal source of food processing industry. Increased awareness in food safety needs a convenient, fast and sophisticated method to detect mycotoxins in the food and livestock products.

The Liquid chromatography coupled to tandem mass spectrometry

（为什么要测定毒素

采用什么方法测定，创新性

2. Experimental

2.1. Chemicals and reagents

All standards (T-2 toxin, HT-2, DAS) were purchased from Sigma-Aldrich(St. Louis, MO, USA),and stored at -20℃. Methanol, Acetonitrile, formic acid, ammonium acetate were HPLC grade purchased from Merck (Darmstadt, Germany).Milli-Q quality water (Millipore, Billerica, MA, USA) were used throughout the whole process. Acetone, ethyl acetate, Methanol, Acetonitrile were of analytical grade were from . charinert Nylon Syringe Filters (0.22µm pore size, 13 mm diameter) were obtained from Agela Technologies.

2.2. Preparation of standard solutions

Stock solution of T-2 toxin (1mg/ml) ,HT-2 toxin (1mg/ml) and DAS (1mg/ml) were prepared in pure acetonitrile, stored at -20℃ in the brown glass vials. Working solutions (100 µg/ml) of each mycotoxin were prepared in acetonitrile . Combined working solution of 1ng/ml, 5ng/ml and 50ng/ml of individual toxins were prepared by mixing a suitable dilution of the three working solutions. All the working solution were stored at 4℃ in the amber glass vials.

2.3. Source of samples

2.3.1. Incubation of Fusarium poae

Fusarium poae (provided from College of Plant Science and Technology, Huazhong Agricultural University ) was isolated from moldy Cereals. T-2 toxin, HT-2 toxin and DAS were the second toxic metabolites of Fusarium poae. In suitable environmental conditions, after 1 week, three primary mycotoxins could be detected in the corn inoculated by Fusarium poae.

2.3.2 Animal experiments

The corn contaminated by Fusarium poae mixed into healthy broiler diet in specific proportion. In 1-day-old broiler chicken (Ross-308) fed a diet containing moldy corn for 1 week and then collected tissues from treated chicks after sacrificing. Simultaneously, bank samples were from control 7-day-old broiler. Collected tissues were heart, liver, spleen, lung, kidney, glandular stomach, muscular stomach, small intestine, muscle, bone and brain. The samples were stored at -20℃ until use.

2.4. Sample pretreatment

After levigated into powder in the mortar at liquid nitrogen, the sample was accurately weighed 1 g. The tissue was transferred into 10ml polypropylene centrifuge, and 3ml ethyl acetate was added, followed by a vortex mixing for 30 min. After put into Ultrasonic Cleaners for 30min, the sample was extracted for 15min. followed by a centrifugation step (15 min, 6000× g).The supernatant was removed to another polypropylene centrifuge and 3 ml ethyl acetate was added. The extraction was vortex mixed again and placed into Ultrasonic Cleaners for 30min. The mixture was centrifuged ( 6000 × g )for 15 min, and then the supernatant was removed.

The combined supernatants were transferred to another polypropylene centrIfuge and evaporated using a gentle stream of N2 at 45 ℃. The dry residue was redissolved by 1 ml acetonitrile, followed by vortex mixing (10s) and ultrasound (30s) to mix the content of the tube. At last, the solution was passed through the 0.22µm Nylon Syringe Filters, and stored at -20℃ to analysis.

2.5. LC-MS/MS analysis

The Liquid chromatography coupled to tandem mass spectrometry (TSQ QUANTUM ULTRA, Thermo Scientific, Brookfield, USA) using selected reaction monitoring (SRM) mode was detected T-2, HT-2 and DAS in the different biomatrices. The LC-MS/MS system consisted of a Waters `` separation module with a `` injection loop coupled to a Quantum Ultra trip quadrupole mass spectrometer equipped with an electrospray ionisation probe operating in positive ionisaton mode. Separation was performed on a Thermo Heparation was performed on a Thermo Hypersil Gold column (100mm×2.1mm, 3.0µm) at 35℃ with the flow-rate of 350 µL/min. For the analysis of T-2, HT-2 and DAS, the mobile phase A consisted of 10mM ammonium acetate and 0.50% formic acid in water, while the mobile phase B was Methanol. A linear gradient elution program was applied as follows: initial 70%B, 3.8min 100%, 4.0min 70% and held on for a further 2 min for re-equilibration until the total run time of 6 min. The injection volume was 5 µL and the autosampler tray was set at 4℃.

The MS spectrometer was operated in the positive electrospray ionization(ESI+) mode. The following settings were used for MS/MS conditions: spray voltage:4000V, vaporizer temperature: 300℃, sheath gas pressure: 5 psi, ion sweep gas pressure: 0 psi, aux valve flow: 5arb(arbitrary units), capillary temperature: 270℃, tube lens offset: 90V, source CID collision energy: 0. Date were obtained and processed by Xcalibur software (Thermo Scientific, Broolfield, USA).

2.6. Method validation

2.6.1. Selectivity and specificity

To evaluate whether or not obvious interference at the retention time in blank samples in the same operating conditions, study of selectivity and specificity of the method was performed. We analyzed difference between the target mycotoxins and interference by comparing chromatograms of blank tissue homogenates and tissue homogenates spiked with T-2, HT-2 and DAS.

2.6.2. Matrix effect, extraction recovery and process efficiency

The matrix effect (ME) of the target analytes by tissue matrix components was measured by comparing the peak areas obtained from the extract solution of blank tissue spiked with three mycotoxins (A) with the peak areas of standard solution containing an equivalent amount of the target mycotoxin (B). Experiments were performed at three levels, 1, 5, 50 ng/ml, in triplicate. The ratio ( A / B × 100% ) was used to evaluate the matrix effect. The value of matrix effect in the standard solution containing the analytes was considered to be 100%. When the value was less than 100%, it indicated that the tissue matrix components showed an ion suppression effect. In contrast, if the value was greater than 100%, the matrix components could enhance ion effect[9].

Extraction recovery (RE) of the analytes was demonstrated by comparing the peak areas obtained from the blank tissue to which three mycotoxins was added before extraction (C) with the peak areas of the same operation above after extraction process. (A) The ratio ( C / A × 100% ) evaluated the extraction recovery. Process efficiency (PE) represented the combination of matrix effect and extraction recovery of the target analytes[10]. It indicated directly the efficiency of the total extraction method. It was evaluated using the following equation: PE (%)=( C / B ) ×100%. Both of the two index were performed at three levels, 1, 5, 50 ng/ml, in triplicate.

2.6.3. Limit of detection ( LOD ) and Limit of quantification ( LOQ )

The limit of detection ( LOD ) was the lowest concentration of the spiked blank samples that could be determined with a signal-to-noise (S/N) ratio of ≥3.The limit of quantification ( LOQ ) was defined the concentration with a signal-to-noise ratio (S/N) of ≥10. The LOD and LOQ of different blank tissues spiked with the same analyte were detected with distinct value.

2.6.4. Intra-day and inter-day precision

The precision was investigated at concentrations of 1 and 50 ng/ml. To estimate intra-day precision, five times of each tissue Homogenate were analyzed on the same day. For inter-day precision, five aliquots of each tissue Homogenate were detected on five continuous days. Precision of the total method in terms of repeat-ability was evaluated on the basis of coefficient of variation (R.S.D(%)).

Accuracy was computed as R.E. (%). (R.E. (%) = (DC-TC)/TC × 100, DC represented the detected concentration and TC represented the theoretical concentration )

2.6.5. Stability

The stability of analytes in chicken tissues was estimated in triplicate at high QC concentration ( 50ng/ml ). The stability experiment included: (A) to evaluated room temperature stability, operated tissue sample was assessed after storage for 8h at room temperature; (B) for autosampler stability, the treated sample was placed in the autosampler for 24h before injected.[9] (C) the experiment of freeze/thaw stability was performed by freeze (-20℃)-thaw (room temperature) for three times. The sample was frozen for 12h and storaged at room temperature for 1h. (D) to investigate long-term stability, the treated samples were detected by the mass spectrometry after storaged at -20 ℃ for at least 2 weeks.

3. Results and discussion

Optimization of detection condition

Optimization of chromatography

Optimization of mass spectrometry

3.2 Sample pretreatment

3.2.1. Optimization of extraction solvents.

To obtain the high process efficiency, four kinds of common solvents were used for former liquid-liquid extraction procedure, including Acetone, ethyl acetate, Methanol, Acetonitrile (84:16). Process efficiency could demonstrated directly the effect of the extraction procedure. Fig.2 illustrated that

In this work, ethyl acetate is most suitable for extracting in the several extraction solvents. ```

3.2.2.Extraction recovery(RE).

Liquid-liquid extraction of analyte using ethyl acetate, yielded high recoveries, mostly ranging from 68% to 110% for spiked all of the tissues containing 1, 5, 50 ng/ml three target toxins. However, there were few lower recoveries of the HT-2 at low level in some biometrics, such as, liver (60.07%), small intestine (60.98%), bone (58.49%). The results were summarized in Table 1. For the different combinations of matrices, the RE of mycotoxin was generally lower in minimum level samples than it in higher level samples except for T-2 in spleen and DAS in small intestine.

3.2.3.Matrix effect(ME).

In this study, the matrix effect (ME) was determined in the low (1ng/mL), middle (5ng/mL) and high (50ng/mL) QC analytes according to the procedure described by Liu Changhui et al[9]. As we know, the Liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS) was specificity and selectivity, but it was also shown that matrix components may affect the ionization efficiency[11]. According to Liquid-liquid extraction of ethyl acetate, this phenomenon had no obvious effect for biometrics analysis. The results were showed in Table 2. For T-2 toxin and DAS the ME values were higher than 61% in the most compounds except for T-2 in lung at low level. Simultaneously, the average ME values of HT-2 toxin were low in some biological matrices, such as liver(51.53%), glandular stomach(53.29%), brain(43.51%) and muscle(40.73%).

3.2.4.Process efficiency.

Process efficiency (PE) indicated the actual toxin concentration of the detected samples. The PE values were calculated by combination of RE and ME of the target biometrics[10]. As a result, either low RE values or low ME values led to imperfect PE data. It was significant to improve extraction rate for ideal test results. The results of PE were summarized in Table 3. For minimum level RE and ME values in low level biological samples, it was showed growth trend from low-dose to high-dose in PE process results. For most biometrics, PE values ranged from 50% to 120% in three target toxins. Nevertheless, imperfect data also appeared in some tissues for HT-2 determination at concentration of 1ng/ml. The PE values were 24.87%, 30.58%, 31.42%, 24.14% and 31.96% in liver, kidney, glandular stomach, muscle and bone, respectively. No matter how High extraction recovery value could not change this phenomenon caused by low matrix effect.

3.3 Limit of detection ( LOD ) and Limit of quantification ( LOQ )

The method detectability was demonstrated by the limit of detection(LOD) and limit of quantification(LOQ) for toxin determination. LOD and LOQ were calculated based on signal-to-noise ratios of 3 / 1 and 10 / 1, respectively, and then the results were showed in table 4. For instance, the LOD values ranged from 0.02ng/ml to 0.05ng/ml for all the compounds in detected samples, and the calculated LOQ values ranged from 0.08ng/ml to 0.15ng/ml in the three analytes. The LOD and LOQ values were much lower than that reported by Baere et al. for the analysis of T-2(i.e. LOD=0.06ng/ml ) and HT-2(i.e. LOD=0.07ng/ml) in chicken plasma.[11] And the detectability of T-2 was similar to the LOD reported by Zou et al. in chicken muscle(i.e. LOD=0.007ng/ml).[12] But no results for the determination of T-2 and HT-2 in another chicken tissues could be found in the journal, demonstrating the novelty of this study. As a result, the presented method was sensitive enough to evaluate the mycotoxins in eleven kinds of biometrics. (空白样品与加标样品的比较)

3.4 Intra-day and inter-day precision

The precision data for intra-day (n=5) and inter-day (n=5) biological samples showed in Table 5 were processed at maximum and minimum levels. The assay values for both occasions (intra- and inter-day) were found to be within the accepted variable limits. The intra- and inter-day precisions (R.S.D(%)) in most biometrics were less than 13.5% and 11.7% for T-2 toxin, 10.8% and 14.5% for HT-2, 9.5% and 10.9% for DAS, respectively. The high assay values were presented at low QC sample concentration. This results suggested that the proposed method could reproducibly assay target compounds in broiler tissues.

3.5 Stability

We investigated the stability of target toxin at high concentration in pretreated samples kept under the different conditions, namely, room temperature for 8 h, in the autosampler for 24h, frozen-thaw for three times and long-term stability (-20℃ for half a month). According to the results of Table 6, no significant degradation was observed in different process above.