Method for enriching, separating and purifying marine fat-soluble scallotoxin-2

Abstract

The invention belongs to the technical field of marine biotoxin standard substance preparation and marine drug development, and particularly relates to a marine fat-soluble scallotoxin-2 enrichment, separation and purification method. Specifically, from the middle ten days of July to the middle ten days of August every year, adsorption bags containing macroporous resin are repeatedly put into a northern sea area and then taken out to enrich scallopoxin-2 in seawater; removing impurities from the enriched scallotoxin-2 solution by using an organic solvent, concentrating, preparing the concentrated solution by using mass spectrum guided liquid chromatography, and separating and purifying the scallotoxin-2 from a sample with multiple impurities and low toxin content; and then preparing by adopting an ultraviolet-guided liquid chromatography, and further separating out the scallotoxin-2 with high purity and high recovery rate. According to the method, enrichment and indoor separation and purification are carried out from seawater, a large amount of scallotoxin-2 with the purity being 99% or above can be obtained, and raw material supply is guaranteed for toxin solution standard substance development and medicine development.

Description

Technical Field

[0001] This invention belongs to the field of marine biological toxin standard material preparation and marine drug development technology, specifically involving a method for enrichment, separation and purification of marine fat-soluble scallop toxin-2. Background Technology

[0002] Pectenotoxin-2 (PTX) is a lipid-soluble marine biotoxin, readily soluble in organic solvents such as ethanol. CAS No.: 97564-91-5, structural formula as follows, molecular formula: C 47 H 70 O 14 Molecular weight: 859.07 g / mol.

[0003]

[0004] Pectenotoxins (PTXs) are a group of complex toxins with numerous isoforms. Currently, 24 components have been identified, with PTX2 being the most common. PTX-1 and PTX-6 can interconvert with PTX4 and PTX-7, and all can be converted into PTX-8 and PTX-9. Scallop toxins are produced by fin algae (…). Dinophysis Scallop toxins (spp.) are produced and distributed in coastal waters worldwide. Marine filter-feeding shellfish can accumulate toxic microalgae in their bodies after ingesting them. The most easily accumulated toxin is scallop toxin-2, which can harm human health. The European Union sets the safe consumption limit for scallop toxin-2 at 0.16 mg / kg, while my country currently lacks a safe consumption limit standard for this toxin. Although scallop toxins are toxic, recent research indicates they have potential value for the development of anti-cancer and other drugs.

[0005]

[0006] my country is a major maritime power, ranking first in the world in seafood production, trade, and consumption. However, with the increasing global warming, ocean acidification, and the growing discharge of land-based organic pollutants into the ocean, coupled with an imbalance in nitrogen and phosphorus nutrient ratios, global marine biotoxin pollution is intensifying. In recent years, scallop toxin-2 has been frequently detected in animals in my country's coastal waters, occasionally exceeding EU standards; it is also frequently detected in imported seafood. Scallop toxin-2 is an important type of marine biotoxin, currently detected using liquid chromatography-tandem mass spectrometry. Therefore, obtaining high-purity scallop toxin-2 is crucial for the development of its standard substances and drug research, ensuring the safe consumption of seafood and the deep utilization of marine biological resources in my country. Scallop toxin-2 is produced by dinoflagellates. Although these toxins easily accumulate in marine bivalves and other marine organisms under natural conditions, the amounts remain extremely small, making field collection insufficient to meet the demand for producing sufficient quantities of high-purity toxins. The algae that produce these toxins are difficult to cultivate under artificial conditions. However, by enriching them at sea, sufficient raw materials can be provided to obtain a sufficient amount of high-purity toxins for the development of standard substances and drugs. Summary of the Invention

[0007] The purpose of this invention is to provide a method for enriching, separating and purifying marine fat-soluble scallop toxin-2.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for enriching, separating, and purifying marine lipid-soluble scallop toxin-2 involves repeatedly placing adsorption bags containing macroporous resin into northern sea areas from mid-July to mid-August each year, and then removing them to enrich scallop toxin-2 in seawater. The enriched scallop toxin-2 solution is then purified by removing impurities and concentrating it with an organic solvent. The concentrate is then prepared by mass spectrometry-guided liquid chromatography to separate and purify scallop toxin-2 from samples with low toxin content. Finally, ultraviolet-guided liquid chromatography is used to further separate and purify scallop toxin-2 with a purity exceeding 99% and a high recovery rate.

[0009] The process involves repeatedly placing absorbent bags from mid-July to mid-August each year, soaking them for 6-8 days each time. The absorbent bags are hung on floating racks on the sea surface, at a depth of 0.2-1.5m below the sea surface.

[0010] Each adsorption bag was rinsed with fresh water to remove impurities. The large-pore resin was then loaded into a chromatography column and eluted with 95% ethanol at a flow rate of 1000 mL / h. The eluent was collected until scallop toxin-2 was no longer detectable, thus enriching scallop toxin-2. The eluent was then vacuum concentrated into an aqueous solution at a low temperature of 10℃-15℃ and stored at -20℃ for later use.

[0011] The resin, after being eluted with ethanol, is soaked in 5% hydrochloric acid and 5% sodium hydroxide for 2 hours each, then rinsed with fresh water until the pH matches that of fresh water. After activation with methanol for 24 hours, it can be reused.

[0012] The enriched scallop toxin-2 (enriched scallop toxin-2 solution eluted with ethanol) was treated with hexane, methanol and acetonitrile, and then vacuum concentrated to a solid at a low temperature of 5℃-10℃. After concentration, scallop toxin-2 was isolated and purified from the concentrated sample with many impurities and low toxin content by mass spectrometry-guided liquid chromatography. Then, scallop toxin-2 with a purity of over 99% and a high recovery rate was further separated by ultraviolet-guided liquid chromatography.

[0013] The scallop toxin-2 eluent (scallop toxin-2 solution eluted with ethanol) was centrifuged to remove the precipitate, then mixed with n-hexane for extraction. After extraction, the n-hexane was concentrated under vacuum to remove it, followed by centrifugation to remove the precipitate. The aqueous solution containing scallop toxin-2 was then concentrated to a solid at a low temperature of 5-10°C. The volume ratio of n-hexane to the centrifuged aqueous solution containing scallop toxin-2 was 1:1. After extraction, the n-hexane was removed by vacuum concentration at 5°C and 30 bar. Anhydrous methanol was added to the above solid sample, the solid sample was dissolved by ultrasound, the precipitate was removed by centrifugation, and the collected centrifuged solution was dark yellow; anhydrous methanol was added to the above centrifuged precipitate, the precipitate was dissolved by ultrasound, and the precipitate was removed by centrifugation, and the solution was light yellow; the methanol solution containing scallop toxin-2 was combined and concentrated to a solid at a low temperature of 5℃-10℃. Anhydrous acetonitrile was added to the above solid sample, and the solid sample was dissolved by ultrasonication. The precipitate was removed by centrifugation, and the solution after centrifugation was dark yellow. Anhydrous acetonitrile was added to the precipitate after centrifugation, and the precipitate was dissolved by ultrasonication. The precipitate was removed by centrifugation, and the solution was light yellow. The acetonitrile solutions containing scallop toxin-2 were combined and concentrated into a liquid at a low temperature of 5℃-10℃ and stored at low temperature.

[0014] After filtration through an organic membrane, the concentrated liquid sample was prepared by mass spectrometry-guided liquid chromatography (MS-guided HPLC). The chromatographic column was C18, the flow rate was 4.8 mL / min, and the mobile phase was water and acetonitrile with gradient elution. The MS-guided HPLC conditions were as follows: the SIM ion concentration of scallop toxin-2 was 876.4, and the sample collected at the elution time of 16.8–17.5 min was scallop toxin-2. The prepared solutions containing scallop toxin-2 were combined and concentrated into liquid under vacuum and stored at -20 °C for later use.

[0015] The sample was collected by mass spectrometry-guided liquid chromatography (MS / LC) and then subjected to UV-guided liquid chromatography (LC). The chromatographic column was C18, the flow rate was 4.8 mL / min, and the mobile phase was water and acetonitrile. The liquid containing the toxin was obtained by UV-guided LC and concentrated into a solid, thus obtaining scallop toxin-2 with a purity of over 99% and a high recovery rate. The UV-guided LC conditions were: UV absorption wavelength of 230 nm, and the sample collected at the peak time of 16.8–17.5 min was scallop toxin-2.

[0016] The mobile phase is water and acetonitrile in a volume percentage ratio of 95-10%:5-90%, wherein the water contains 2 mM ammonium formate and 50 mM formic acid, and the acetonitrile is an aqueous solution of acetonitrile containing 2 mM ammonium formate and 50 mM formic acid.

[0017] Furthermore, the above mobile phases were prepared using mass spectrometry-guided liquid chromatography. Mobile phase A consisted of an aqueous solution of 2 mM ammonium formate and 50 mM formic acid, and mobile phase B consisted of an aqueous solution of acetonitrile containing 2 mM ammonium formate and 50 mM formic acid. From 0 to 1 minute, the acetonitrile content increased from 5% to 10%; from 1 to 15 minutes, it increased from 10% to 90%; from 15 to 20 minutes, it reached 90%; from 20 to 23 minutes, it decreased from 90% to 10%; and from 23 to 25 minutes, it decreased from 10% to 5%. The sample collected at the peak time of 16.8–17.5 minutes was scallop toxin-2. The eluent containing scallop toxin-2 was combined and concentrated into a liquid under vacuum at low temperature, in a volume not exceeding 50 mL (i.e., one test tube), for later use.

[0018] In the separation process of this invention, since scallop toxin-2 has a low ability to absorb ultraviolet light, mass spectrometry-guided liquid chromatography with high sensitivity is preferred to purify scallop toxin-2 from samples with many impurities and low content as much as possible, thereby obtaining scallop toxin-2 with a purity of over 99% and a high recovery rate.

[0019] Technical advantages of the present invention: This invention enriches, removes impurities, and purifies scallop toxin-2 at sea, achieving a toxin purity of over 99% and a yield exceeding 50 mg, thus ensuring a reliable supply of raw materials for the development of toxin standard substances and drug development. The technical advantages are: (1) By deploying large-pore resin in natural sea areas from mid-July to mid-August in northern waters to adsorb scallop toxin-2, large quantities of scallop toxin-2 can be obtained, solving the problem of toxin raw material source. (2) Hexane, methanol, and acetonitrile are used to remove impurities from the scallop toxin-2 sample, followed by low-temperature vacuum concentration and drying, significantly improving the recovery rate of scallop toxin-2 compared to conventional rotary evaporation and freeze-drying methods. (3) Scallop toxin-2 has weak ultraviolet absorption. In the preparation of the primary phase with high impurity content and low concentration, it is difficult to use an ultraviolet spectroscopy detector as a guide for toxin collection. Therefore, mass spectrometry is used to collect the sample, effectively removing impurities and concentrating scallop toxin-2 in the sample. When the scallop toxin 2 is prepared and concentrated in one step with fewer impurities and a high concentration, using an ultraviolet detector as a guide for sample collection can significantly improve the preparation efficiency and increase the recovery rate. Attached Figure Description Figure 1 This is a flowchart of the process for enriching, separating, and purifying scallop toxin-2 according to the present invention.

[0020] Figure 2 This is a weekly chart showing the annual variation of macroporous resin scallop toxin-2 enrichment in natural marine areas according to the present invention.

[0021] Figure 3 The graph shows the recovery rate of scallop toxin-2 in vacuum concentrated ethanol at different temperatures and vacuum levels (10℃, 15 bar; 15℃, 25 bar; 20℃, 35 bar; 25℃, 45 bar; 30℃, 55 bar) at different times.

[0022] Figure 4 The graph shows the recovery rate of scallop toxin-2 in water under different temperatures and vacuum conditions (5℃, 10 bar, 10℃, 20 bar, 15℃, 30 bar, 20℃, 40 bar, 25℃, 50 bar) at different times during vacuum concentration and drying according to the present invention.

[0023] Figure 5 This is a liquid phase ultraviolet purity detection spectrum of scallop toxin-2 according to the present invention.

[0024] Figure 6 This is the liquid chromatography-tandem mass spectrometry (LC-MS / MS) spectrum of scallop toxin-2 in this invention. Detailed Implementation

[0025] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0026] Example 1 The specific steps for enriching scallop toxin-2 from seawater are as follows: Figure 1 As shown: (1) Adsorption bags were made by sewing polyester mesh fabric with a diameter of 4cm, a length of 90cm, and a pore size of 48μm. Each bag was filled with about 80g of macroporous resin (Mitsubishi Chemical (China) Trading Co., Ltd. macroporous resin HP20, which needs to be activated with methanol for 24 hours before use) (wet weight). The adsorption bags were hung on floating racks in the coastal waters of northern my country. The floating racks were constructed according to existing technology and located 0.2 to 1.5m below the sea surface (1.0m in this example). The enrichment efficiency of scallop toxin-2 was significantly higher in mid-July to mid-August (weeks 28 to 32) than in other seasons. Figure 2 From mid-July (week 28) to mid-August (week 32), macroporous resin can be added multiple times to enrich scallop toxin-2 in seawater (in this example, 200 scallops are added each time, spaced 1 meter apart, for 5 times). After each addition and soaking for 7 days, the enrichment bags are removed, and the bags are rinsed with fresh water to remove impurities. The macroporous resin is then loaded into a chromatography column (5 cm in diameter, 80 cm in length glass frit), and eluted with 95% ethanol at a flow rate of 1000 mL / h. Each column is washed with 10 liters of 95% ethanol. After this process, scallop toxin-2 is no longer detectable in the eluent, which is then ready for use.

[0027] The resin, after being eluted with ethanol, is then soaked in 5% hydrochloric acid and 5% sodium hydroxide for 2 hours each, rinsed with fresh water until the pH matches that of fresh water, and then activated with methanol for 24 hours for reuse.

[0028] (2) The above-mentioned scallop toxin-2 aqueous solution (yellow aqueous solution) eluted with ethanol was concentrated to remove ethanol within a different temperature range of 10-30°C and under a vacuum of 15-55 bar (see Figure 3 ),Depend on Figure 3 It is evident that the lower the temperature, the higher the scallop toxin recovery rate, but the process is time-consuming. Therefore, in order to achieve high yields, the process is carried out at low temperatures.

[0029] Example 2 The specific steps for the isolation and purification of scallop toxin-2 are as follows: Figure 1 As shown: (1) As shown in Figure 1, take 50L of the collected scallop toxin-2 elution concentrated aqueous solution, centrifuge to remove the precipitate at 10,000 rpm (Toshiba CR22N high-speed centrifuge) for 30 minutes. After centrifugation, the collected liquid is mixed with n-hexane and extracted at a ratio of n-hexane: collected liquid after centrifugation of 1:1 (v / v). After the separatory funnel is placed in a refrigerator at 4℃ for 1 hour, it is vacuum concentrated (Buchi R 300 rotary evaporator) at 10℃ and 30 bar to remove n-hexane, and then centrifuged to remove the precipitate at 10,000 rpm for 15 minutes. Then the aqueous solution containing scallop toxin-2 is concentrated to solid at 5℃-25℃ under vacuum. Figure 4 ), Depend on Figure 4 It is evident that the lower the temperature, the higher the scallop toxin recovery rate, but the process is time-consuming. In order to achieve high yields, the process is carried out at low temperatures of 5 or 10°C.

[0030] (2) Anhydrous methanol was added to the solid sample in (1) above, and the solid sample was dissolved by ultrasonication. The precipitate was removed by centrifugation at 10,000 rpm for 15 minutes. The solution after centrifugation was dark yellow. Anhydrous methanol was added to the collected precipitate again, and the precipitate was dissolved by ultrasonication. The precipitate was then removed by centrifugation under the above conditions. The solution was light yellow. The methanol solution containing scallop toxin-2 was combined and concentrated to a solid state at 5°C and 12 bar vacuum.

[0031] (3) Add anhydrous acetonitrile to the solid sample in (2) above, dissolve the solid sample by sonication, remove the precipitate by centrifugation at 10,000 rpm for 15 minutes. The solution after centrifugation is dark yellow. Add anhydrous acetonitrile to the collected precipitate, dissolve the precipitate by sonication, remove the precipitate by centrifugation, and the solution is slightly yellow. Combine the acetonitrile solutions containing scallop toxin-2, concentrate them to no more than 50 mL of liquid at 5 °C and 12 bar vacuum, and store at -20 °C.

[0032] (4) Mass spectrometry-guided liquid chromatography purification of toxins. The concentrated sample from (3) above was filtered through a 0.22 μm organic membrane and then prepared by mass spectrometry-guided liquid chromatography (Agilent HPLC 1260 + MS6120 mass spectrometer detector). The chromatographic column was C18 (10×250 mm, 5 μm), the flow rate was 4.8 mL / min, and the mobile phase A was an aqueous solution of 2 mM ammonium formate and 50 mM formic acid. The mobile phase B was an aqueous solution of acetonitrile containing 2 mM ammonium formate and 50 mM formic acid. The gradient of water and acetonitrile in the liquid mobile phase is shown in Table 1. Specifically, from 0 to 1 minute, the acetonitrile ratio increased from 5% to 10%; from 1 to 15 minutes, the acetonitrile ratio increased from 10% to 90%; from 15 to 20 minutes, the acetonitrile ratio was 90%; from 20 to 23 minutes, the acetonitrile ratio decreased from 90% to 10%; and from 23 to 25 minutes, the acetonitrile ratio decreased from 10% to 5%. Samples collected at peak elution times of 16.8–17.5 minutes were identified as scallop toxin-2. The eluents containing scallop toxin-2 were combined and analyzed by liquid chromatography-tandem mass spectrometry (AB SCIEX, API4000) to determine the toxin content in each tube. The resulting solutions containing scallop toxin-2 were then collected and concentrated to a volume not exceeding 50 mL under vacuum at 5 °C and 12 bar. The solutions were then stored at -20 °C for later use.

[0033] Table 1 Preparation of liquid mobile phase gradient

[0034] (5) Purification of toxins by UV-guided liquid chromatography. Samples prepared by mass spectrometry-guided liquid chromatography were prepared using a UV-guided liquid chromatograph (Agilent HPLC 1260 + UV detector DAD VL). The column was C18 (10×250mm, 5 μm), the flow rate was 4.8 mL / min, the injection volume was 100 μL, and the mobile phase was the same as in step (4). The UV-guided liquid chromatography-mass spectrometry conditions were: UV absorption wavelength of 230 nm. Samples collected at elution times of 16.8–17.5 minutes were scallop toxin-2. Vacuum concentration was performed. If the purity did not reach 99%, the process was repeated once more under the UV-guided liquid chromatography conditions. After 1–2 preparations, the liquid containing scallop toxin-2 was collected and dried under vacuum at 5°C and 12 bar. The solid powder was white.

[0035] The above-mentioned white solid powder scallop toxin-2 (see...) Figure 5 and 6 The purity was first tested using liquid chromatography-ultraviolet light detector. After exceeding 99%, it was verified using liquid chromatography-tandem mass spectrometry (AB SCIEX, API4000). The results showed that the purity of scallop toxin-2 was 99.8% and the weight was 58 mg. The purity and quantity fully met the requirements for the preparation of standard scallop toxin-2 solution.

Claims

1. A method for enriching and purifying a marine fat-soluble pectenotoxin-2, characterized by comprising the steps of: The adsorption bag filled with macroporous resin is repeatedly put into the northern sea area from mid-July to mid-August every year, and then taken out to realize the enrichment of saxitoxin-2 in seawater; then the enriched saxitoxin-2 solution is treated with an organic solvent to remove impurities and concentrated, and then the concentrated solution is prepared by mass spectrometry-guided liquid chromatography to separate and purify saxitoxin-2 from a sample with low toxin content; then the ultraviolet-guided liquid chromatography preparation is used to further separate and purify the saxitoxin-2 with a purity of more than 99% and a high recovery rate. ​ 2. The method for enriching and purifying oceanic fat-soluble pectenotoxin-2 according to claim 1, characterized in that: The adsorption bag is repeatedly put into the sea area from mid-July to mid-August every year, and soaked for 6-8 days each time; wherein the adsorption bag is hung on the sea surface float, and located at a position 0.2-1.5 m below the surface of seawater.

3. The method for enriching and purifying a marine fat-soluble pectenotoxin-2 according to claim 1 or 2, characterized by: The adsorption bag put each time is washed with fresh water to remove sundries on the bag, the macroporous resin is loaded into a chromatography column, eluted with 95% ethanol at a flow rate of 1000 ml / h, and the eluate is collected until saxitoxin-2 is not detected to realize the enrichment of saxitoxin-2, and then concentrated into a liquid at a low temperature of 10-15°C under vacuum.

4. The method for enriching and purifying ocean fat-soluble pectenotoxin-2 according to claim 3, characterized in that: The resin eluted with ethanol is soaked with 5% hydrochloric acid and 5% sodium hydroxide for 2 hours respectively, then washed with fresh water until the pH is consistent with that of fresh water, and then activated with methanol for 24 hours to be reusable.

5. The method for enriching and purifying a marine fat-soluble pectenotoxin-2 according to claim 1, characterized by: The enriched saxitoxin-2 is treated with n-hexane, methanol and acetonitrile, and concentrated into a solid at a low temperature of 5-10°C under vacuum, and then separated and purified from a sample with low toxin content and many impurities by mass spectrometry-guided liquid chromatography preparation after concentration; and then further separated by ultraviolet-guided liquid chromatography preparation to obtain saxitoxin-2 with a purity of more than 99% and a high recovery rate.

6. The method for enriching and purifying ocean fat-soluble pectenotoxin-2 according to claim 5, characterized in that: The saxitoxin-2 eluted and concentrated solution obtained in claim 3 is centrifuged to remove the precipitate, mixed with n-hexane for extraction, and then concentrated under vacuum to remove n-hexane after extraction, and then the precipitate is removed by centrifugation again, and the aqueous solution containing saxitoxin-2 is concentrated into a solid at a low temperature of 5-10°C; wherein the volume ratio of n-hexane to the aqueous solution containing saxitoxin-2 collected by centrifugation is 1:1; and the n-hexane is removed by vacuum concentration under the condition of 5°C and 30 bar after extraction; Anhydrous methanol is added to the above solid sample, the solid sample is dissolved by ultrasonic, the precipitate is removed by centrifugation, and the solution after centrifugation is dark yellow; anhydrous methanol is added to the above precipitate after centrifugation, the precipitate is dissolved by ultrasonic, and then the precipitate is removed by centrifugation again, and the solution is light yellow; the methanol solution containing saxitoxin-2 is combined and concentrated into a solid at a low temperature of 5-10°C. Anhydrous acetonitrile is added to the above solid sample, the solid sample is dissolved by ultrasonic, the precipitate is removed by centrifugation, and the solution after centrifugation is dark yellow; anhydrous acetonitrile is added to the above precipitate after centrifugation, the precipitate is dissolved by ultrasonic, and then the precipitate is removed by centrifugation again, and the solution is light yellow; the acetonitrile solution containing saxitoxin-2 is combined and concentrated into a liquid at a low temperature of 5-10°C, and then stored at a low temperature.

7. The method for enriching and purifying marine fat-soluble pectenotoxin-2 according to claim 5, characterized by: The concentrated liquid sample is filtered through an organic membrane, and then subjected to mass spectrometry-guided liquid chromatography preparation, a C18 column, a flow rate of 4.8 mL / min, a mobile phase of water and acetonitrile, gradient elution, and mass spectrometry-guided liquid chromatography conditions: the SIM ion of saxitoxin-2 is 876.4, and the sample collected at the peak time of 16.8-17.5 min is saxitoxin-2; the preparation liquid containing saxitoxin-2 is collected and combined, concentrated into a liquid under vacuum, and then stored at -20℃ for use.

8. The method for enriching and purifying marine fat-soluble pectenotoxin-2 according to claim 5, characterized by: The liquid sample collected by mass spectrometry-guided liquid chromatography is subjected to ultraviolet-guided liquid chromatography preparation, a C18 column, a flow rate of 4.8 mL / min, a mobile phase of water and acetonitrile, and ultraviolet-guided liquid chromatography preparation to obtain a liquid containing toxins, which is concentrated into a solid to obtain saxitoxin-2 with a purity of more than 99% and a high recovery rate; The ultraviolet-guided liquid chromatography conditions are: the ultraviolet absorption wavelength is 230 nm, and the sample collected at the peak time of 16.8-17.5 min is saxitoxin-2.

9. The method for enriching and purifying marine fat-soluble pectenotoxin-2 according to claim 7 and 8, characterized in that: The mobile phase is water and acetonitrile at 95-10%:5-90% by volume percentage, wherein the water contains 2 mM ammonium formate and 50 mM formic acid, and the acetonitrile is a 2 mM ammonium formate and 50 mM formic acid acetonitrile solution.

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