Rapid detection method for PSTs and TTX substances in human plasma

Abstract

The present invention discloses a method for rapidly determining PSTs and TTX substances in human plasma, the method comprising the following steps: (1) taking a plasma sample, adding an acetonitrile solution precipitant containing trichloroacetic acid, performing protein precipitation, and obtaining a filtrate; (2) activating a hydrophilic solid phase extraction plate, balancing, loading the filtrate, rinsing, and eluting to obtain an eluted eluate; (3) performing LC-MS / MS analysis on the eluate eluted in step (2) to determine the content of PSTs and TTX substances. The method of the present invention can be used for qualitative and quantitative detection of PSTs and TTX substances in human plasma, with reliable results, low detection limit, and fast speed.

Description

Technical Field

[0001] The invention belongs to the field of biological analysis, and in particular relates to a method for rapidly determining paralytic shellfish toxins and tetrodotoxin-like substances in human plasma. Background Art

[0002] Tetrodotoxins mainly include tetrodotoxin (TTX), 11-oxTTX, 11-deoxyTTX, 11-norTTX-6(R)-ol, 11-norTTX-6(S)-ol, 4-epiTTX, 4,9-anhydroTTX, 5,6,11-trideoxyTTX, 4-CysTTX, 5-deoxyTTX, 5,11-dideoxyTTX and 6,11-dideoxyTTX. They are highly toxic non-protein neurotoxins widely found in pufferfish and some amphibians. The degree of poisoning from TTX is related to the amount of intake, the time after intake and the health status before poisoning. As a class of potent toxins, it can selectively block sodium ion (NaV) channels, such as NaV1.1, NaV1.2, NaV1.3, NaV1.6 and NaV1.7. These channels are widely distributed in the human central and peripheral nervous systems. They achieve nerve paralysis by blocking the conduction of nerve impulses and are considered to be promising pain treatment drugs. Among them, TTX is the most studied. It has been reported that the lethal dose of TTX for humans is estimated to be 6-7 μg / kg body weight, and blood pressure will tend to decrease at 0.5 μg / kg body weight. When TTX is used in clinical treatment, its dose is far lower than LD50 (LD50 for intraperitoneal injection in mice is 8 μg / kg). The current clinical trial report of the dosage is 15-90 μg (60 kg / person), and the Cmax in the blood is about 1 ng / mL.

[0003] Paralytic shellfish toxins (PSTs) are a group of natural toxic alkaloids, mainly including: Saxitoxin (STX), Neo-Saxitoxin (neo-STX), Decarbamoyl saxitoxin (dc-STX), Gonyautoxin 1-6 (GTX 1-6), N-Sulfocarbamoylgonyautoxin 2 (C1), N-Sulfocarbamoylgonyautoxin 3 (C2), Decarbamoyl gonyautoxin 2 (dc-GTX 2), Decarbamoylgonyautoxin 3 (dc-GTX 3), etc. It is mainly derived from toxic algae in red tides. Its toxicity is similar to that of tetrodotoxins. Both can selectively block sodium ion channels, causing nerve paralysis and even respiratory paralysis. The range of human poisoning caused by PSTs is between 600 and 5000 μM, and the lethal dose is 3000 to 30000 μM. According to the similarity of the groups, it can be divided into four categories: carbamate toxins, N-sulfonamide formyl toxins, decarbamate formyl toxins and deoxyformyl toxins. In recent years, it has been found that it and TTX-like substances are often detected in shellfish. Therefore, in order to ensure the safety of patients and understand the relationship between drug concentration and efficacy, a sensitive, accurate and reliable detection method is urgently needed to simultaneously monitor the levels of TTX-like substances and PSTs in human blood.

[0004] At present, the methods for determining TTX-like substances and PSTs include mouse biological method, liquid fluorescence method, enzyme-linked immunosorbent assay, and liquid chromatography-tandem mass spectrometry. However, the mouse biological method requires detection of specific animals, and the pretreatment is cumbersome and has poor repeatability; although the liquid fluorescence method is easy to operate, it has low sensitivity and poor specificity. The enzyme-linked immunosorbent assay has high sensitivity and strong specificity, but the pretreatment is complex, the reaction is time-consuming, and the operation is difficult. Compared with the previous methods, liquid chromatography-tandem mass spectrometry is the most advantageous technology. It has the characteristics of good specificity and high sensitivity. It can more quickly and accurately determine the concentration of TTX-like substances and PSTs in human blood, thereby providing a more efficient and accurate basis for clinical diagnosis. However, this technology still has great challenges, mainly due to the poor enrichment effect and poor interference removal ability of the pretreatment method.

[0005] TTX and PSTs are both highly polar alkaline substances with poor retention on traditional reversed-phase columns and low mass spectrometry ionization efficiency. Hydrophilic interaction liquid chromatography (HILIC) can help solve these problems and show good retention and ionization efficiency in LC-MS. The suppression of target analyte ions (matrix effect) caused by the co-elution matrix in biological samples is another challenge for the quantitative detection of these two substances in plasma. The most commonly used technology for the preparation of samples of such substances is solid phase extraction (SPE). Based on the structural characteristics of TTX and PSTs, three fillers with different mechanisms of action may be used, one of which is cation exchange extraction. Because the positively charged groups in the compound structure interact strongly with the sulfonic acid groups of the stationary phase, the target analyte is firmly retained on SPE, and only strong acids such as hydrochloric acid can elute it. The sample eluted from SPE needs solvent evaporation and redissolution to remove the strong acid before entering LC-MS detection, which is time-consuming and laborious, and usually other impurities may be introduced due to the addition of strong acid. Therefore, strong cation exchange extraction technology is currently rarely used for the detection of such substances. Another SPE material is a graphitized carbon carrier, which uses the reverse phase adsorption principle to retain the target analyte. Due to the high polarity of the compound, the recovery rate is usually poor. The main reason is that the stationary phase has limited ability to retain it and the ion suppression caused by insufficient matrix separation. Due to the hydrophilicity of the target analyte, hydrophilic interaction-solid phase extraction (HILIC-SPE) can be used as a third option. HILIC-SPE is a process that uses hydrophilic interactions to retain hydrophilic compounds on the filler and then elutes them. However, the HILIC force is not strong, so the retention capacity and the ability to remove impurities are also limited. In order to meet the recovery rate and impurity removal effect at the same time, some people use C18 and HILIC SPE fillers successively to increase the enrichment and impurity removal of such substances. This method includes two SPE extraction processes, and the eluted product needs to be blown dry at 90°C. The steps are time-consuming and relatively complicated [Fong BM et al. Talanta. 2011 Jan 15; 83 (3): 1030-6.]. Currently, there is no simple, accurate and sensitive detection method for TTX and PSTs in plasma samples.

[0006] Therefore, there is an urgent need in the art for a simple, rapid, accurate and sensitive detection method for determining TTX-like substances and PSTs. Summary of the invention

[0007] The purpose of the present invention is to provide a simple, rapid, accurate and sensitive method for detecting TTX substances and PSTs.

[0008] Therefore, the present invention investigates three commercially available HILIC-SPE plates (silanol SPE, amide SPE, and polycarboxylic acid SPE), innovates the composition of pretreatment reagents, changes the single interaction force between TTX-like substances and PSTs and the HILIC-SPE stationary phase, so that the two have a dual mode of hydrophilic interaction and ion exchange when combined. This technology is used for the pretreatment of plasma samples and combined with LC-MS / MS to detect TTX-like substances and PSTs in plasma. The method has good sensitivity, small matrix effect, and high recovery rate.

[0009] The present invention provides a method for rapidly determining PSTs and TTX-like substances in human plasma, characterized in that the method comprises the following steps:

[0010] (1) Take a plasma sample and add an acetonitrile solution containing trichloroacetic acid as a precipitant, which contains the internal standard Arg- 15 N4, after protein precipitation, obtain the filtrate;

[0011] (2) activating a hydrophilic solid phase extraction plate with methanol and balancing it with acetonitrile, loading the filtrate obtained in step (1) onto the hydrophilic solid phase extraction plate, rinsing it once with 95% acetonitrile, and then adding an eluent of trichloroacetic acid-acetonitrile solution for elution;

[0012] (3) The eluate eluted in step (2) is subjected to LC-MS / MS analysis to determine the content of PSTs and TTX-like substances.

[0013] The mass percentage concentration of trichloroacetic acid in the acetonitrile solution containing trichloroacetic acid in the above step (1) is 0.5%-10%, and the internal standard Arg- 15 The mass percentage concentration of N4 is 0.1-10 ng / ml. Preferably, the internal standard Arg- 15 The mass percentage concentration of N4 is 0.71ng / ml.

[0014] In the above step (2), the solid phase extraction plate is a hydrophilic solid phase extraction plate containing a polycarboxylic acid bonded phase, a silanol bonded phase and an amide bonded phase. Preferably, the solid phase extraction plate is a hydrophilic solid phase extraction plate containing a polycarboxylic acid bonded phase, which is also referred to as a polycarboxylic acid SPE herein.

[0015] The trichloroacetic acid-acetonitrile solution in the above step (3) is composed of: 0.5%-5% trichloroacetic acid by mass percentage, 40%-60% acetonitrile by mass percentage, and the rest water.

[0016] In a specific embodiment, the trichloroacetic acid-acetonitrile solution in the above step (3) is composed of 1% by weight trichloroacetic acid, 50% by weight acetonitrile, and the remainder water.

[0017] The above-mentioned PSTs are one or more of saxitoxin, neosaxitoxin, decarbamoyl saxitoxin, anacardin 1 to 6, N-sulfonylcarbamoyl anacardin 2, N-sulfonylcarbamoyl anacardin 3, decarbamoyl anacardin 2 or decarbamoyl anacardin 3.

[0018] The above-mentioned TTX-type substances include one or more of tetrodotoxin, 11-oxTTX, 11-deoxyTTX, 11-norTTX-6(R)-ol, 11-norTTX-6(S)-ol, 4-epiTTX, 4,9-anhydroTTX, 5,6,11-trideoxyTTX, 4-CysTTX, 5-deoxyTTX, 5,11-dideoxyTTX or 6,11-dideoxyTTX.

[0019] The above-mentioned PSTs are toxins from the cyanobacteria, and TTX-like substances are tetrodotoxins.

[0020] In the above step (1), positive pressure is applied to the sedimentation plate to obtain the filtrate.

[0021] The mass percentage concentration of trichloroacetic acid in the trichloroacetic acid acetonitrile solution in the above step (1) is 0.5%-10%. Preferably, the mass percentage concentration of trichloroacetic acid in the trichloroacetic acid acetonitrile solution is 0.5%-5%.

[0022] The present invention establishes an SPE extraction technology based on HILIC-ion exchange dual action mode to process plasma samples for LC-MS / MS method detection of TTX-like substances and PSTs in human plasma.

[0023] A. The present invention adds trichloroacetic acid reagent to the processing solvent, optimizes the composition of the processing solvent, changes the traditional single HILIC retention principle to a HILIC-ion exchange dual action mode, greatly enhances the interaction between the stationary phase and polar and alkaline compounds, and improves the recovery rate of TTX substances and PSTs;

[0024] B. The trichloroacetic acid reagent used in the present invention, in addition to increasing the retention of TTX substances and PSTs on SPE, is also a component of the precipitant. This precipitant containing the trichloroacetic acid reagent increases the dissociation of the analyte from plasma proteins, thereby facilitating the release of TTX substances and PSTs in plasma so that they can better interact with SPE and further improve the recovery rate;

[0025] C. The present invention increases the retention of TTX-like substances and PSTs on SPE, while removing the co-elution of some polar and non-polar interfering substances that often interfere with their detection in the past, so that the plasma sample has no obvious matrix effect after treatment;

[0026] D. The treatment method described in the present invention is combined with LC-MS / MS to detect TTX-like substances and PSTs in plasma, with high recovery rate, good accuracy and precision, and a linear range that can well meet the concentration range of pharmacokinetic research and clinical poisoning of TTX-like substances and PSTs. The detection time for one injection is 4.5 minutes, the analysis time is short, and the pre-treatment does not require evaporation and concentration, which is simple and fast.

[0027] (1) Release of TTX-like substances and PSTs: Different from other protein precipitation methods, in this method, plasma needs to be precipitated and released using an organic solvent precipitant containing trichloroacetic acid, thereby increasing the protein dissociation degree and release rate of TTX-like substances and PSTs.

[0028] (2) Composition of the loading solution: This method investigated the effects of four loading solutions on the recovery rates of TTX-like substances and PSTs. After comparison, it was found that the acid-containing loading solution had stronger retention, and the loading solution containing trichloroacetic acid was stronger than formic acid. It was speculated that the acid-containing loading solution could enable TTX-like substances and PSTs to form HILIC-ion exchange dual-mode SPE with HILIC-SPE, thereby increasing the recovery rate. The type and concentration of the acid may result in different strengths of the interaction.

[0029] (3) Selection of SPE plates: Different from traditional HILIC-SPE or simple ion exchange mode SPE, the three HILIC-SPEs selected in this method are partially converted into ionic states in the loading solution containing acid (trichloroacetic acid), which can produce different degrees of ion exchange effects on TTX and PSTs, thereby producing different degrees of retention of TTX and PSTs. Among them, polycarboxylic acid SPE has the strongest retention of TTX and PSTs, followed by silanol SPE, and finally amide SPE.

[0030] Beneficial effects of the present invention:

[0031] Compared with previously published methods, this method has the following advantages:

[0032] A. The present invention adopts a rapid precipitation combined with SPE process, without the steps of drying and reorganization, and is easy to operate and has good reproducibility. The LC-MS / MS detection time is only 4.5 minutes, which greatly saves time cost and is conducive to automated conversion.

[0033] B. The present invention can detect extremely trace concentrations of TTX-like substances and PSTs in plasma samples, with a quantitative lower limit of 0.1 ng / mL, providing clinically highly sensitive detection results.

[0034] C. This method has good accuracy, high precision, good recovery, wide linear range, and no obvious matrix effect. It has excellent performance and is suitable for the pharmacokinetics and toxicity studies of TTX-like substances and PSTs in the human body.

[0035] D. The extraction mechanism of the dual-mode SPE pretreatment method using HILIC-ion exchange for detection in liquid chromatography-tandem mass spectrometry has not been reported. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Recovery rates of three SPE plates screened by TTX and GTX5;

[0037] Figure 2 TTX and GTX5 investigated the recovery rates of different loading solutions;

[0038] Figure 3 TTX linear range diagram;

[0039] Figure 4 GTX5 linear range graph;

[0040] Figure 5 Chromatogram of each substance in the sample.

[0041] a. Chromatogram of TTX in LLOQ sample and blank sample (TTX 0.1 ng / mL);

[0042] b. Chromatogram of GTX5 in LLOQ sample and blank sample (GTX5 0.1 ng / mL);

[0043] c. Chromatogram of internal standard in LLOQ sample and blank sample (internal standard Arg- 15 N45ng / mL). DETAILED DESCRIPTION

[0044] The present invention is described in more detail and specifically with reference to the following examples, but the following examples are not intended to limit the present invention, and the reagents of the present invention can be purchased on the market. The percentage concentrations of the present invention are all mass percentage concentrations unless otherwise specified.

[0045] Example 1 Detection of TTX and GTX5

[0046] (1) Plasma sample pretreatment

[0047] ①Whether to add acid to the loading solution and comparison of different acids

[0048] 100 μL of plasma sample to be tested was drawn into a 96-well collection plate, and then 700 μL of precipitant (acetonitrile, acetonitrile solution containing formic acid or trichloroacetic acid, both containing internal standard Arg- 15N40.71ng / mL), put the 96-well collection plate on an oscillator and vortex mix for 5 minutes at 1200rpm, then transfer all the mixed samples to a 96-well precipitation plate (2ml / well) under positive pressure, take all the filtrate and apply it to SPE (in this case, polycarboxylic acid SPE, purchased from Tianjin Saifeile Biotechnology Co., Ltd., item number: S-iHX960302-W, specification: 30mg / 2mL / well), and the eluate is detected by LC-MS / MS. The recovery rates of different loading solutions are shown in Figure 2 .

[0049] ②Comparison of recovery rates of three HILIC-SPE plates

[0050] Pipette 100μL of the sample to be tested into a 96-well collection plate, and perform the precipitation treatment in the same way as in step ① (the precipitant is 0.5%-5% trichloroacetic acid-acetonitrile solution, and in this case, it is 1% trichloroacetic acid-acetonitrile solution). Take all the filtrate for SPE treatment. This method investigated three types of 96-well SPE plates (silicon hydroxyl SPE, amide SPE, and polycarboxylic acid SPE). After activation and equilibrium with methanol and acetonitrile, the filtrate was loaded into the SPE plate and positively pressurized. After the filtrate was completely dripped into the 96-well collection plate, the waste liquid in the collection plate was discarded, and 200μL of eluent (80%-100% acetonitrile, 95% acetonitrile was used in this case) was added. Positive pressure was applied again, and after the eluent was completely dripped into the 96-well collection plate, the waste liquid in the collection plate was discarded. Add 200 μL of eluent (0.5%-5% trichloroacetic acid 40%-60% acetonitrile solution, this method uses 1% trichloroacetic acid-50% acetonitrile solution, the rest is water) to the SPE plate, wait for the eluent to drip completely into the 96-well collection plate, and finally detect by LC-MS / MS. The recovery rates of the three SPE plates are shown in Figure 1 .

[0051] Combination Figure 1 and Figure 2 It can be seen that the recovery rate of plasma TTX and GTX5 with trichloroacetic acid as the precipitant is the highest, while the recovery rate of TTX and GTX5 by polycarboxylic acid SPE is the highest among the three HILIC-SPEs, which are about 80% and 78%, respectively.

[0052] (2) Liquid phase conditions

[0053]

[0054] (3) Mass spectrometry conditions

[0055]

[0056]

[0057] (4) Implementation results and data analysis

[0058] ①Sensitivity and linear range

[0059] The method has good linearity, and the R values ​​of TTX and GTX5 in the 200-fold linear range (0.1-20 ng / mL) are 0.9997 (see Figure 3 ) and 1.0000 (see Figure 4 ), the lower limit of quantification of TTX (S / N>10) is about 0.1 ng / mL (see Figure 5 a), the lower limit of quantification (S / N>10) of GTX5 is about 0.1 ng / mL (see Figure 5 b).

[0060] ②Accuracy, precision, recovery and matrix effect of the method

[0061] This method investigated the accuracy, precision, recovery and matrix effect of addition, and was validated in three consecutive batches. The results showed (see Table 1 and Table 2) that TTX and GTX5 in plasma had good accuracy and precision within the linear range. The intra-batch accuracy of TTX and GTX5 was 98.50%-99.82% and 96.21%-98.75%, respectively, and the inter-batch accuracy was 98.84%-99.48% and 97.23%-99.48%, respectively. The intra-batch and inter-batch precision of the TTX method was <6% and <7%, respectively, and the intra-batch and inter-batch precision of the GTX5 method was <6% and <6%, respectively. The plasma extraction recoveries of TTX and GTX5 were about 80% and 78%, respectively, with no obvious matrix effect.

[0062] Table 1. Method validation results of TTX in plasma

[0063]

[0064] Table 2. Method validation results of GTX5 in plasma

[0065]

[0066]

[0067] In addition, it should be understood that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that various modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the scope and essence of the present invention.

Claims

1. A method for rapidly determining PSTs and TTX-like substances in human plasma, characterized in that: The method comprises the following steps: (1) Plasma samples were added to a 96-well collection plate, and acetonitrile solution containing trichloroacetic acid was added as a precipitant, containing the internal standard Arg- 15 N4, after protein precipitation, all the samples after precipitation and mixing are transferred to a 96-well precipitation plate under positive pressure to obtain a filtrate; the mass percentage concentration of trichloroacetic acid in the acetonitrile solution of trichloroacetic acid is 0.5%-5%; (2) activating a 96-well hydrophilic solid phase extraction plate with methanol and balancing it with acetonitrile, loading the filtrate obtained in step (1) onto a 96-well hydrophilic solid phase extraction plate containing a polycarboxylic acid bonded phase, rinsing once with a 95% acetonitrile solution, and then eluting with an eluent of a trichloroacetic acid-acetonitrile solution; the trichloroacetic acid-acetonitrile solution is composed of: 0.5% to 5% trichloroacetic acid by mass percentage, 40% to 60% acetonitrile by mass percentage, and the remainder water; (3) The eluate eluted in step (2) is subjected to LC-MS / MS analysis to determine the content of PSTs and TTX-like substances.

2. The method according to claim 1, characterized in that The mass percentage concentration of trichloroacetic acid in the acetonitrile solution precipitant containing trichloroacetic acid in step (1) is 0.5%-10%, and the internal standard Arg- 15 The mass percentage concentration of N4 is 0.1-10ng / ml.

3. The method according to claim 1, characterized in that The trichloroacetic acid-acetonitrile solution in step (2) is composed of 1% by weight of trichloroacetic acid, 50% by weight of acetonitrile, and the remainder of water.

4. The method according to any one of claims 1 to 3, characterized in that: The PSTs are one or more of saxitoxin, neosaxitoxin, decarbamoyl saxitoxin, agatoxins 1 to 6, N-sulfonylcarbamoyl agatoxin 2, N-sulfonylcarbamoyl agatoxin 3, decarbamoyl agatoxin 2 or decarbamoyl agatoxin 3.

5. The method according to any one of claims 1 to 3, characterized in that: The TTX-type substances include one or more of tetrodotoxin, 11-oxTTX, 11-deoxyTTX, 11-norTTX-6(R)-ol, 11-norTTX-6(S)-ol, 4-epiTTX, 4,9-anhydroTTX, 5,6,11-trideoxyTTX, 4-CysTTX, 5-deoxyTTX, 5,11-dideoxyTTX or 6,11-dideoxyTTX.

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