CN112979666A - Terramycin derivative, preparation method and detection method thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了如结构式

所示的土霉素衍生物，其制备方法以及检测其含量的方法，本发明利用多种高效液相制备色谱对土霉素水解物进行分离纯化，制得2种土霉素水解衍生物M390‑1和M390‑2，并对水样中土霉素水解衍生物的含量进行定量测定，确定其在水样中的浓度。为研究两种土霉素衍生物在环境样品中的浓度水平，评价土霉素对环境的影响提供准确的评估依据，进一步提高土霉素相关产品的质量标准，提高其安全性和可控性。The present invention discloses a structural formula such as

The oxytetracycline derivative shown, its preparation method and the method for detecting its content, the present invention utilizes a variety of high performance liquid phase preparative chromatography to separate and purify the oxytetracycline hydrolyzate to obtain two kinds of oxytetracycline hydrolyzed derivatives M390 ‑1 and M390‑2, and quantitatively determine the content of oxytetracycline hydrolyzed derivatives in water samples to determine their concentrations in water samples. In order to study the concentration levels of two oxytetracycline derivatives in environmental samples, provide an accurate assessment basis for evaluating the impact of oxytetracycline on the environment, further improve the quality standards of oxytetracycline-related products, and improve their safety and controllability .

Description

Terramycin derivative, preparation method and detection method thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a hydrolysate of tetracycline antibiotics (oxytetracycline), and a preparation method, a separation and purification method and an analysis and detection method of the hydrolysate.

Background

Oxytetracycline (OTC) is in the form of pale yellow tablets or sugar-coated tablets, is a tetracycline, and can be used for treating rickettsia including epidemic typhus, endemic typhus, rocky mountain fever, tsutsugamushi disease and Q fever, mycoplasma infection and chlamydia infection. Terramycin has broad-spectrum anti-pathogenic microorganism effect, is a rapid bacteriostatic agent, and has bactericidal effect on certain bacteria at high concentration. The action mechanism is that the medicine can be specifically combined with the A position of ribosome 30S subunit to prevent the connection of aminoacyl-tRNA at the position, so that the growth of peptide chain is inhibited and the protein synthesis of bacteria or other pathogenic microorganisms is affected. The terramycin has stronger antibacterial activity on staphylococcus aureus, pneumococcus, streptococcus pyogenes, gonococcus, meningococcus, escherichia coli, aerobacter, shigella, yersinia, listeria monocytogenes and the like; in addition, terramycin also has strong effects on rickettsia, mycoplasma, chlamydia, actinomycetes and the like. Terramycin has good therapeutic effect on respiratory tract infection and intestinal tract infection. The effect of terramycin on controlling amebic enteritis and intestinal tract infection is superior to that of tetracycline, doxycycline, methacycline, minocycline and the like. The oxytetracycline for animals needs a large amount, and the curative effect of treating the swine fever and swine enzootic pneumonia is better than that of tetracycline. The antibiotics have good effects on preventing and treating certain livestock and poultry diseases and promoting the growth of animals. The structure of oxytetracycline is as follows.

Hydrolysis is known to be one of the major chemical transformation pathways for drug degradation in the environment, and both temperature and pH are important factors affecting hydrolysis. Many antibiotics, such as tetracycline, penicillin, erythromycin and sulfonamides, are poorly stable in aqueous solutions and are susceptible to hydrolysis to form epimers and dehydration products. There are studies on the establishment of a forced hydrolysis process (85 ℃, pH7) for oxytetracycline to remove it rapidly. At present, few research reports are carried out on terramycin hydrolysate, and only 3 kinds of known and existing standard hydrolysate (EOTC (oxytetracycline), alpha-apo-OTC (alpha-adriamycin) and beta-apo-OTC (beta-adriamycin)) are three antibacterial activity conversion/degradation products of terramycin and are identified in different culture media in the terramycin degradation process. Weakly acidic and neutral solutions are particularly beneficial for the formation and/or accumulation of EOTC, which may be converted back to oxytetracycline or directly further degraded. Acidic and basic pH conditions are particularly favorable for the formation of α -apo-OTC, with β -apo-OTC being detected in lesser amounts at all pH conditions. Further degradation to ter-OTC (irreversible process) may occur for α -apo-OTC, β -apo-OTC, but this requires severe acidic conditions. OTC may contain another unique impurity, 2-acetyl-2-decaformamido-oxytetracycline (ADOTC), present as a fermentation by-product.

As the research on terramycin related products is early, but the research on terramycin hydrolysis related to the prior art does not deeply explore the hydrolysis products of terramycin, and the mechanism of the rapid degradation of terramycin in the intensified hydrolysis is not known. The existing research only probes the hydrolysis effect of the terramycin, but does not detect and research the composition of a terramycin hydrolysate and the qualitative and quantitative content of the components of the product, and the quantitative research on the concentration level of the hydrolysate of the sewage after the terramycin is produced in the environment is less, especially the separation and purification of a terramycin water product; there are fewer studies on the concentration of its hydrolysate as an assessment of environmental risk.

Disclosure of Invention

The invention aims to solve the technical problems in the quantitative research of the components of terramycin (OTC) hydrolysate, particularly the components of sewage treatment product after terramycin production and the content of the hydrolysate, and provides terramycin hydrolysate, a preparation method and a detection method thereof, the invention provides two terramycin hydrolysis derivatives, two brand-new products M390-1 and M390-2 are found in the terramycin hydrolysis process by utilizing ultra-high liquid chromatography-quadrupole tandem time-of-flight mass spectrometry (UPLC-QTOF/MS), and the two products are separated and purified by utilizing various high-efficiency preparative chromatographs, so that pure products (the purity reaches 98 percent and is more than 99 percent respectively) are obtained, and further M390-1 and M390-2 substances in various water samples can be quantitatively detected to determine the specific concentration thereof, the concentration levels of these two newly discovered substances in environmental samples were explored to achieve a better assessment of environmental risk.

To achieve the object of the present invention, in one aspect, the present invention provides a oxytetracycline derivative M390-1 of structural formula (i):

in another aspect, the present invention provides a terramycin derivative M390-2 according to formula (II):

the invention also provides a method for preparing terramycin derivatives M390-1 and M390-2, which comprises the steps of firstly carrying out hydrolysis treatment on terramycin to prepare a terramycin hydrolysis solution; and then separating and purifying the oxytetracycline hydrolysis solution by adopting a high performance liquid chromatography.

In still another aspect, the present invention provides a process for the preparation of oxytetracycline derivatives M390-1, M390-2, comprising the steps of:

1) injecting the oxytetracycline hydrolysis solution or oxytetracycline production wastewater into a medium-pressure high-performance liquid preparation chromatograph, performing first purification treatment (namely medium-pressure purification treatment), and collecting eluent according to retention time to obtain first purified liquid;

2) concentrating the first purified solution, injecting the concentrated first purified solution into a high-pressure high-performance liquid preparative chromatograph for second purification treatment, and collecting eluent according to retention time to respectively obtain first characteristic peak purified solution and second characteristic peak purified solution;

3) concentrating the first characteristic peak purified solution, injecting the concentrated first characteristic peak purified solution into a high-pressure high-performance liquid preparative chromatograph for third purification treatment, and collecting eluent according to retention time to obtain a terramycin derivative M390-1;

4) and concentrating the second characteristic peak purified solution, injecting the concentrated second characteristic peak purified solution into a high-pressure high-performance liquid preparative chromatograph for fourth purification treatment, and collecting eluent according to retention time to obtain the oxytetracycline derivative M390-2.

Wherein, the eluent with the retention time of 13-15.8min is collected in the step 1) to obtain the first purified liquid.

In particular, the mobile phases of the first purification treatment are: a is 0.05% formic acid solution, B is 0.05% methanol.

In particular, the flow rates of the mobile phases are: 90 ml/min; the detection wavelength of the first purification treatment is as follows: 210nm and 254 nm;

in particular, the first purification treatment is carried out with a gradient elution, and the gradient elution procedure is: 0-16.8 min: a 80% → 69.8%; 16.8-17 min: a 69.8% → 0%; 17-23 min: and A is 0 percent.

Wherein the pressure is controlled to be less than or equal to 150psi, preferably 90-150psi, during the first purification treatment (i.e. the medium-pressure purification treatment).

In particular, the flow rate of the first purification treatment is 90mL/min, operating at room temperature (20-25 ℃).

In particular, the model of the medium-pressure high performance liquid preparative chromatograph is Agela HP-FALSH HS-1000T.

In particular, the column selection of the medium-pressure high performance liquid preparation chromatograph: WelFlash C18-I column, preferably WelFlash C18-I (20-40um,330 g).

In particular, the method further comprises mass spectrometry of the first purified solution, wherein m/z of a substance contained in the first purified solution is 390.1189.

Particularly, the mass spectrometry is carried out by adopting a QTOF/MS instrument, and the mass spectrometry conditions are as follows:

an ion source: electrospray ion source (ESI); the scanning mode is as follows: a positive ion mode; and (3) monitoring mode: MSE; capillary voltage: 2.5 kV; taper hole gas flow: 60L/h; source temperature: 150 ℃; desolventizing gas temperature: 500 ℃; desolventizing agent gas flow: 600L/h; scanning time: 0.2 s; scanning range: 50-600 Da; collision energy voltage: 15-30V.

Wherein the oxytetracycline hydrolysis solution in the step 1) is prepared by the following method: and (3) carrying out hydrolysis reaction on the oxytetracycline solution at the temperature of 80-90 ℃ for at least 3 h.

Particularly, the method also comprises the steps of adding a sodium tetraborate buffer solution into the oxytetracycline solution, adjusting the pH of the mixed solution to 7, and then carrying out the hydrolysis reaction.

In particular, the concentration of the sodium tetraborate buffer is 5-15mM, preferably 10 mM; the pH of the buffer was 7.

The sodium tetraborate buffer serves to maintain the pH of the reaction mixture throughout the hydrolysis reaction and to stabilize the pH of the reaction mixture at about 7, since oxytetracycline degrades most rapidly at pH 7.

In particular, the sodium tetraborate buffer is formulated as follows: mixing and dissolving sodium tetraborate with ultrapure water, adding hydrochloric acid to adjust the pH to 7, and preparing a sodium tetraborate buffer solution with the concentration of 5-15mM and the pH of 7.

In particular, the sodium tetraborate buffer has a concentration of 10mM and a pH of 7. 3.82g of sodium tetraborate are added per 1000ml of buffer solution.

Precisely weighing a sodium tetraborate solution (3.82g) in a 1000mL volumetric flask, adding ultrapure water to 1000mL, fully dissolving, pouring into a beaker, and adding hydrochloric acid to adjust the pH value to 7 to obtain the sodium tetraborate.

Wherein, the concentration of the OTC solution is 800-1200mg/L, preferably 1000 mg/L.

In particular, the OTC solution is prepared as follows: OTC and ultrapure water are mixed and dissolved to prepare an OTC solution with the concentration of 800-1200mg/L, preferably 1000 mg/L.

In particular, the volume ratio of the OTC solution to sodium tetraborate buffer is 1: 8-10, preferably 1: 9.

in particular, the hydrolysis reaction temperature is preferably 85 ℃; the hydrolysis reaction time is preferably 3 to 9 hours, and more preferably 3 to 8 hours.

In order to prevent the OTC from generating photolysis reaction in the hydrolysis reaction process, shading paper such as aluminum foil paper is used for shading in the process of carrying out hydrolysis reaction by heating in water bath.

In particular, a sodium tetraborate buffer solution is added to the oxytetracycline solution, and the pH of the mixed solution is adjusted to 7 with sodium hydroxide.

Particularly, the oxytetracycline production wastewater is wastewater generated in each stage in the oxytetracycline production process.

The oxytetracycline production wastewater is oxytetracycline pharmaceutical industry wastewater generated in the process of producing oxytetracycline through the working procedures of separation, purification, refining and the like after the oxytetracycline is produced.

Collecting eluent with retention time of 16.8-18min in the step 2) to obtain the first characteristic peak purified liquid; collecting the eluent with the retention time of 19-20min to obtain the second characteristic peak purified liquid.

In particular, the mobile phase of the second purification treatment is: a is 0.05% formic acid solution, B is methanol.

In particular, the mobile phase flow rates are: 100 ml/min; the detection wavelength of the second purification treatment is as follows: 210nm and 254 nm;

in particular, a gradient elution is performed during the second purification treatment and the gradient elution procedure is: 0-22 min: a 80% → 77%; 22-22.5 min: a 77% → 0%; 22.5-27 min: and A is 0 percent.

Wherein the pressure in the second high-pressure purification treatment process is controlled to be 2.5-3.4 MPa.

In particular, the flow rate during the second purification treatment is 100mL/min, operating at room temperature (20-25 ℃).

In particular, the high pressure hplc preparative chromatograph is model BRIX 1860.

In particular, the column selection of the high pressure high performance liquid preparative chromatograph: an Ultimate XB-C18 column, preferably Ultimate XB-C18(250 x 50mm 10 μm).

Particularly, the method further comprises concentrating the first purified solution to a solid, dissolving the solid in a diluent to prepare a first purified concentrated solution, and then performing the second purification treatment.

In particular, the diluents are: methanol: ultrapure water 1:1 (v/v).

Particularly, the collected first and second characteristic peak purified liquids are subjected to mass spectrometric detection, and the m/z of a substance in the characteristic peak purified liquid is 390, so that the first and second characteristic peak purified liquids with the m/z of 390 are obtained. The first characteristic peak purified liquid is M390-1 purified liquid; the second characteristic peak purified liquid is M390-2 purified liquid.

Wherein, eluent with retention time of 26-28min is collected in the step 3), concentrated and dried to obtain the oxytetracycline derivative M390-1.

In particular, the mobile phase of the third purification treatment is: a is ultrapure water, and B is methanol.

In particular, the mobile phase flow rates are: 100 ml/min; the detection wavelength of the second purification treatment is as follows: 210nm and 254 nm;

in particular, a gradient elution is performed during the third purification treatment and the gradient elution procedure is: 0-30 min: a 95% → 70%; 30-30.5 min: a 70% → 0%; 30.5-35 min: and A is 0 percent.

Wherein the pressure in the third high-pressure purification treatment process is controlled to be 2.5-3.4 MPa.

In particular, the flow rate of the third purification treatment is 100mL/min, operating at room temperature (20-25 ℃).

In particular, the high pressure hplc preparative chromatograph is model BRIX 1860.

In particular, the column selection of the high pressure high performance liquid preparative chromatograph: an Ultimate XB-C18 column, preferably Ultimate XB-C18(250 x 50mm 7 μm).

Particularly, the method further comprises the third purification treatment after the first characteristic peak purified liquid is concentrated into a solid and then dissolved by a diluent to prepare the first characteristic concentrated liquid.

In particular, the diluents are: methanol: ultrapure water 1:1 (v/v).

The purified liquid after the second purification treatment is only a crude product and has low purity, so a high-pressure high-efficiency liquid preparation phase chromatograph is required to be further used for purification to remove other impurities. And (3) feeding the eluent of the first characteristic peak after the second purification treatment into a high-pressure high-efficiency liquid preparation phase chromatograph to obtain M390-1, wherein the retention time of the M390-1 is 26-28min, and performing multiple separation to obtain M390-1 purified liquid with higher purity.

Wherein, eluent with retention time of 22-24min is collected in the step 4), concentrated and dried to obtain the oxytetracycline derivative M390-2.

In particular, the mobile phase of the fourth purification treatment is: a is ultrapure water, and B is methanol.

In particular, the mobile phase flow rates are: 100 ml/min; the detection wavelength of the second purification treatment is as follows: 210nm and 254 nm;

in particular, a gradient elution is performed during the fourth purification treatment and the gradient elution procedure is: 0-24 min: a 90% → 70%; 24-24.5 min: a 70% → 0%; 24.5-28 min: and A is 0 percent.

Wherein, the pressure is controlled to be 2.5-3.4MPa in the fourth high-pressure purification treatment process.

In particular, the fourth purification treatment is carried out at a flow rate of 100mL/min and at room temperature (20-25 ℃).

In particular, the high pressure hplc preparative chromatograph is model BRIX 1860.

In particular, the column selection of the high pressure high performance liquid preparative chromatograph: an Ultimate XB-C18 column, preferably Ultimate XB-C18(250 x 50mm 7 μm).

Particularly, the method further comprises the fourth purification treatment after the second characteristic peak purified liquid is concentrated into a solid and then dissolved by a diluent to prepare a second characteristic concentrated liquid.

In particular, the diluents are: methanol: ultrapure water 1:1 (v/v).

The invention also provides a method for determining the contents of oxytetracycline derivatives M390-1 and M390-2 in oxytetracycline waste water, which comprises the following steps:

A) preparing solutions for M390-1 and 390-2 standard curves with the concentrations of 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01mg/L by using oxytetracycline derivative M390-1 and 390-2 standard samples respectively;

B) precisely absorbing the solutions for the M390-1 and M390-2 standard curves respectively, injecting the solutions into a high performance liquid chromatograph respectively, performing liquid chromatography determination, and determining and recording chromatographic peaks, retention times and peak areas corresponding to the oxytetracycline derivatives M390-1 and M390-2 respectively under the concentrations in the step A);

C) respectively drawing standard curves of M390-1 and M390-2 by taking peak areas of chromatographic peaks of oxytetracycline derivatives M390-1 and M390-2 as ordinate and concentration as abscissa, wherein a linear regression equation of the oxytetracycline derivative M390-1 is as follows: y1 ═ 19.854X 1-535.49; wherein Y1 is the peak area of M390-1, and X1 is the concentration of M390-1; the linear regression equation of the terramycin derivative M390-2 is as follows: y2 is 55.731X2-1250.8, wherein Y2 is the peak area of M390-2, and X2 is the concentration of M390-2;

D) pretreating terramycin wastewater to prepare a detection diluent, then performing LC-MS/MS analysis detection, comparing with the retention time of standard M390-1 and M390-2 respectively, and recording and determining chromatographic peaks and peak areas of M390-1 and M390-2 in the terramycin wastewater corresponding to the retention time of the standard M390-1 and M390-2 respectively;

E) respectively substituting the peak areas of M390-1 and M390-2 measured in the step D) into corresponding linear regression equations to calculate the concentrations of M390-1 and M390-2.

Wherein, the terramycin derivatives M390-1 and 390-2 standard substance in the step A) is prepared according to the preparation method of the terramycin derivatives M390-1 and 390-2.

Wherein the chromatographic conditions of the liquid chromatography determination in step B) are as follows: mobile phase: a is 0.1% formic acid solution, B is methanol; gradient elution procedure: 0-1 min: a95%; 1-4.5 min: a 95% → 75%; 4.5-7 min: a 75% → 10%; 7-7.5 min: a 10% → 95%; 7.5-8.5 min: and A95%.

In particular, the mobile phase flow rate: 0.3 ml/min; column temperature: 35 ℃; sample temperature: 10 ℃; sample introduction amount: 10 μ L.

In particular, the retention time of M390-1 and M390-2 is respectively as follows: 6.26min, 6.49 min.

Particularly, the solutions for respectively and precisely absorbing the M390-1 and M390-2 standard curves can be respectively injected into a high performance liquid chromatography mass spectrometer (UPLC-MS/MS) for chromatographic and mass spectrometric determination, and chromatographic peaks, retention times and peak areas corresponding to the oxytetracycline derivatives M390-1 and M390-2 respectively under the concentrations in the step A) are respectively determined and recorded;

in particular, the mass spectrometry conditions are as follows: an ion source: electrospray ion source (ESI); the scanning mode is as follows: a positive ion mode; and (3) monitoring mode: multiple reaction detection mode (MRM); capillary voltage: 2.5 kV; taper hole voltage: 30V; source temperature: 150 ℃; desolventizing gas temperature: 500 ℃; desolventizing agent gas flow: 600L/h; taper hole gas flow: 60L/h.

Wherein, the detection diluent in the step D) is prepared according to the following steps:

D1) filtering oxytetracycline production wastewater by adopting a 0.45-micron water-phase filter membrane, adding ultrapure water and a metal chelating agent into filtrate, and adjusting the pH to 3 by using dilute hydrochloric acid or sodium hydroxide solution to obtain a filtered sample solution;

D2) performing solid phase extraction treatment on the filtered sample liquid, eluting with methanol in the solid phase extraction process, drying the elution effluent liquid, and dissolving with methanol to obtain a solid phase extraction-concentrated sample liquid; then adding ultrapure water, and diluting to obtain a detection diluent.

Wherein the volume ratio of the filtrate to the added ultrapure water in the step D1) is 1: (6-20), preferably 1: 10.

In particular, the metal chelating agent is selected from Na₂EDTA (disodium ethylenediaminetetraacetate) or EDTMPA (ethylenediaminetetramethylenephosphonic acid), preferably Na₂EDTA。

In particular, the ratio of the mass of the added metal chelating agent to the volume of the filtrate is 0.05-0.2: 50(m/v), preferably 0.1:50, i.e. 0.05-0.2g of metal chelating agent per 50ml of filtrate.

Taking 50mL of filtered wastewater sample, respectively adding ultrapure water to 500mL, adding Na₂EDTA (0.1g) is used as a metal chelating agent to remove the interference of metal ions in the wastewater, dilute hydrochloric acid or sodium hydroxide solution is used for adjusting the pH value to 3, and then solid phase extraction is carried out;

wherein, the solid phase treatment in the step D2) is to load the filtered sample liquid into a solid phase extraction column and then rinse the solid phase extraction column with ultrapure water; then drying the loaded solid phase extraction column by using nitrogen; followed by methanol elution and the elution effluent was collected.

Particularly, the solid phase extraction column for the solid phase extraction treatment is an Oasis HLB solid phase extraction column.

In particular, the method further comprises activating the solid phase extraction column, namely washing the solid phase extraction column with dichloromethane, methanol and phosphate buffer solution (pH 3.0) in sequence.

In particular, after the eluate is dried, the dried residue is just dissolved with an appropriate amount of methanol (usually 0.5ml) to obtain the solid phase extraction-concentrated sample solution.

Specifically, the ratio of the volume of the ultrapure water to the volume of the solid-phase extraction-concentration sample liquid is 1:1 (V/V).

Particularly, the elution effluent in the step D2) is concentrated to prepare a solid phase extraction-concentrated sample solution, and then ultrapure water is added to prepare a detection diluent.

In particular, the volume of the assay diluent is typically 1-2ml, preferably 1 ml.

Wherein, in the LC-MS/MS analysis and detection process of the step D), the chromatographic conditions are as follows: mobile phase: a is 0.1% formic acid solution, B is methanol; gradient elution.

In particular, the gradient elution procedure in the LC-MS/MS analysis and detection process is as follows: 0-1 min: a95%; 1-4.5 min: a 95% → 75%; 4.5-7 min: a 75% → 10%; 7-7.5 min: a 10% → 95%; 7.5-8.5 min: and A95%.

In particular, the LC-MS/MS analysis detects that the chromatographic column: waters ACUITY UPLC BEH (1.7 μm, 2.1 mm. times.100 mm); flow rate of mobile phase: 0.3 ml/min; column temperature: 35 ℃; sample temperature: 10 ℃; sample introduction amount: 10 μ L.

Wherein, in the LC-MS/MS analysis and detection process of the step D), the mass spectrum conditions are as follows: an ion source: electrospray ion source (ESI); the scanning mode is as follows: a positive ion mode; and (3) monitoring mode: multiple reaction detection mode (MRM).

In particular, capillary voltage during mass spectrometry: 2.5 kV; taper hole voltage: 30V; source temperature: 150 ℃; desolventizing gas temperature: 500 ℃; desolventizing agent gas flow: 600L/h; taper hole gas flow: 60L/h.

For harmless treatment of antibiotic terramycin, it is far from insufficient to degrade the parent body only to reduce the concentration of the antibiotic terramycin in the environment, and the hydrolysis process cannot completely mineralize the parent terramycinIs CO₂And H₂O, what substances are produced by the degradation of terramycin after the hydrolysis reaction, and the characteristics of various degradation products such as toxicity, titer and the like generated by the degradation need to be deeply researched, so that the waste after the production of terramycin is harmless to the environment or the harm degree of the waste is reduced as much as possible. In order to deeply explore the relevant hydrolysis products of the oxytetracycline, the invention adopts UPLC-QTOF/MS to carry out full-scan analysis on the whole process of the oxytetracycline hydrolysis process, finds two new products from the whole process, further separates and purifies the new products, establishes a UPLC-MS/MS detection method, and simultaneously has corresponding detection in the actual sewage treatment plant samples.

The invention provides two new OTC hydrolysis products, namely oxytetracycline derivatives M390-1 and M390-2; separating and purifying OTC hydrolysate to obtain M390-1 and M390-2; also provides an identification method of M390-1 and M390-2 and a content detection method thereof, which provide basis for understanding and controlling the detection condition of the hydrolysis product and the concentration level thereof in the whole flow of the actual OTC sewage treatment.

According to the invention, UPLC-QTOF/MS is utilized to find two brand-new products M390-1 and M390-2 in the process of terramycin hydrolysis, and simultaneously, a plurality of high-efficiency preparative chromatographs are utilized to separate and purify the two products to obtain pure products M390-1 and M390-2 (the purity is respectively up to 98% and more than 99%), quantitative detection is carried out on M390-1 and M390-2 substances in a plurality of water samples to determine the concentration of the substances, and the concentration levels of M390-1 and M390-2 in environmental samples are explored to achieve better environmental risk evaluation.

The invention has the following beneficial effects:

(1) the invention discovers two novel terramycin hydrolysates which are reported for the first time;

(2) the preparation method and the separation and purification method of the hydrolysate provided by the invention are simple and easy to implement, and the two hydrolysates can be prepared in a large scale;

(3) the UPLC-MS/MS analysis and detection method provided by the invention can rapidly separate and detect the hydrolysate in the oxytetracycline hydrolysis solution, has good selectivity and extremely high sensitivity, has the detection limit as low as 10 mug/L, and can be used for quantitative detection of M390-1 and M390-2 substances in a sample;

(4) the hydrolysate provided by the invention can be used as a standard substance for checking related substances of the oxytetracycline (such as inlet and outlet water of an actual sewage treatment plant) and used for detecting and controlling the content of the degradation product, so that the quality standard of related products of the oxytetracycline is further improved, and the safety and the controllability of the product are improved.

Drawings

FIG. 1 is a total ion flow diagram of a water sample before and after 8h of hydrolysis of OTC detected by UPLC-QTOF/MS, wherein A is before hydrolysis; b is hydrolysis for 8 hours;

FIG. 2 shows the purity results of the separated and purified M390-1 and M390-2 by UV detection, wherein A is M390-1 and B is M390-2;

FIG. 3 is a chromatogram of M390-1 and M390-2 pure substances after separation and purification by UPLC-MS/MS detection;

FIG. 3A is a hydrogen nuclear magnetic resonance spectrum of the product of OTC hydrolysis M390-1;

FIG. 3B is a nuclear magnetic resonance carbon spectrum of the product M390-1 of the OTC hydrolysis;

FIG. 3C is a hydrogen nuclear magnetic resonance spectrum of the product M390-2 of the OTC hydrolysis;

FIG. 3D is a nuclear magnetic resonance carbon spectrum of the product M390-2 of the OTC hydrolysis;

FIG. 4A is a standard graph of OTC hydrolyzate M390-1;

FIG. 4B is a standard graph of OTC hydrolyzate M390-2;

FIG. 5 is a process flow of oxytetracycline wastewater treatment in test example 1;

FIG. 6 is a chromatogram of two hydrolysates (M390-1 and M390-2) detected in the actual wastewater of test example 1 with the respective corresponding standard substances;

FIG. 7 is the concentrations of two hydrolysis products detected in the actual wastewater of test example 1;

FIG. 8 shows a wastewater treatment process in the small test apparatus for oxytetracycline in test example 2;

FIG. 9 is a chromatogram of two hydrolysates (M390-1 and M390-2) detected in the actual wastewater of test example 2 with the respective corresponding standard substances

FIG. 10 is a graph showing the concentrations of two kinds of hydrolysis products detected in the actual wastewater of test example 2.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Using an instrument: ultra-high performance liquid chromatography-electrospray tandem quadrupole mass spectrometry (UPLC-MS/MS), ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-QTOF/MS), medium-pressure high performance liquid preparative chromatograph (Agela HP-FALSH HS-1000T), high-pressure preparative liquid chromatography (BRIX 1860), and the like.

Example 1 identification of oxytetracycline derivatives

1) Sodium tetraborate (Na) is prepared₂B₄O₇·10H₂O) buffer:

precisely weighing sodium tetraborate solution (3.82g) in a 1000mL volumetric flask, adding ultrapure water to 1000mL, fully dissolving, pouring into a beaker, adding hydrochloric acid to adjust pH to 7, and preparing into 10mM sodium tetraborate (Na) with pH of 7₂B₄O₇·10H₂O) a buffer solution;

the pH of the sodium tetraborate solution was adjusted from 9.21 to pH7 by adding hydrochloric acid to the sodium tetraborate solution (sodium tetraborate buffer was used to maintain the pH of oxytetracycline during hydrolysis).

2) Preparing a first OTC solution:

precisely weighing 1g of OTC (oxytetracycline hydrochloride, CAS number: 2058-46-0), placing in a 1000mL brown volumetric flask, adding ultrapure water for dissolving, and preparing into a first OTC solution with the concentration of 1000 mg/L;

3) preparing a second OTC solution:

putting every 50mL of the first OTC solution into a 500mL volumetric flask, adding 450mL of sodium tetraborate buffer (pH7) with pH7 and 10mM, uniformly mixing, pouring into a beaker, and adjusting the pH to 7 by using sodium hydroxide to prepare a second OTC solution, wherein the concentration of the second OTC solution is 100mg/L, and the pH is 7;

the OTC solution was prepared in a brown volumetric flask to prevent photolytic reaction of the OTC.

4) OTC hydrolysis reaction

Pouring the second OTC solution into a glass bottle, heating in a constant-temperature water bath kettle, keeping the temperature of the water bath at 85 ℃ (usually 80-90 ℃), and carrying out hydrolysis reaction for 8h (usually not less than 3h, preferably 7-9h) to obtain OTC hydrolysis reaction solution, sampling at 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 240, 300, 360, 420 and 480min respectively in the hydrolysis reaction process, collecting the mixed solution of the OTC hydrolysis reaction, taking 7-8mL each time, putting the mixed solution into a 10mL centrifuge tube, and sampling after uniformly mixing the hydrolysis reaction solution before sampling each time. And (3) wrapping the sample solution with aluminum foil paper, putting the wrapped sample solution into an ice bath, cooling the wrapped sample solution to normal temperature, measuring the sample by using ultra performance liquid chromatography-time of flight mass spectrometry (UPLC-QTOF/MS), and detecting the type of a hydrolysate generated along with the extension of hydrolysis time in the OTC hydrolysis process.

In order to prevent the OTC from photolysis reaction, attention is paid to shading by shading paper such as aluminum foil paper and the like in the water bath heating reaction process; in addition, in the process of sampling and determining the types of the hydrolysis products, the oxytetracycline is degraded relatively quickly at the beginning of the reaction, so that sampling is carried out at short intervals, and the oxytetracycline is basically completely degraded by about 3 hours, but the sampling is not finished until 8 hours in order to more completely and more comprehensively explore the change of the hydrolysis products in the hydrolysis process.

5) UPLC-QTOF/MS analysis of samples

And (2) carrying out full scanning on the sampled sample in the hydrolysis reaction process by using an UPLC-QTOF/MS instrument, namely carrying out UPLC-QTOF/MS analysis on the sampled sample in the OTC hydrolysis reaction process, wherein:

5-1) chromatographic conditions were as follows:

a chromatographic column: waters ACUITY UPLC BEH (1.7 μm, 2.1 mm. times.100 mm); mobile phase: a is 0.1% formic acid solution, B is methanol; gradient elution procedure: 0-1 min: a95%; 1-4.5 min: a 95% → 75%; 4.5-13 min: a 75% → 10%; 13-15 min: a10 percent; 15-17 min: a 10% → 95%; flow rate of mobile phase: 0.3 ml/min; column temperature: 35 ℃; sample temperature: 10 ℃; sample introduction amount: 10 mu L of the solution;

5-2) Mass Spectrometry conditions were as follows:

an ion source: electrospray ion source (ESI); the scanning mode is as follows: a positive ion mode; and (3) monitoring mode: MSE; capillary voltage: 2.5 kV; taper hole gas flow: 60L/h; source temperature: 150 ℃; desolventizing gas temperature: 500 ℃; desolventizing agent gas flow: 600L/h; scanning time: 0.2 s; scanning range: 50-600 Da; collision energy voltage: 15-30V;

5-3) analyzing and detecting the sample:

the UPLC-QTOF/MS is used for detecting the identification of unknown products in the OTC hydrolysis process, and the total ion flow graph of water samples before and after the OTC hydrolysis is 8h, as shown in figure 1. The hydrolysate is two new substances with M/z being 390.1189, which are named as M390-1 and M390-2 according to the retention time, wherein the retention time of the substance M390-1 is 6.50-6.70 min; the retention time of the substance M390-2 is 7.10-7.30 min.

In the samples sampled at other reaction times, UPLC-QTOF/MS detected several other unknown products, but the peak areas were not considered as important because they were small relative to the two species with m/z of 390.1189. Meanwhile, due to the continuous progress of the hydrolysis reaction, part of unknown products are degraded secondarily after being generated, and the two substances with the m/z being 390.1189 are kept stable and are not degraded secondarily after being generated. The conditions of the chromatogram and the mass spectrum are suitable for identifying the unknown product in the OTC hydrolysis process.

EXAMPLE 2 preparation of oxytetracycline derivatives M390-1 and M390-2

1. First purification treatment

Preparing a second OTC solution (7000ml) according to the method of example 1, and then carrying out hydrolysis reaction in a constant temperature water bath at 85 ℃ (usually 80-90 ℃) for 3h (the hydrolysis reaction time is more than or equal to 3h, preferably 3 h);

dividing the hydrolysis reaction liquid after reacting for 3h into 35 parts, injecting samples for 35 times, injecting 200ml each time, injecting into a medium-pressure high-efficiency liquid preparative phase chromatograph (the model is Agela HP-FALSH HS-1000T), carrying out first purification treatment (namely medium-pressure purification treatment), then collecting eluent with retention time of 13-15.8min to obtain first purification liquid, wherein the first purification liquid collected each time is about 300ml, combining the eluents subjected to 35 times of purification treatment, and keeping 10-11L for later use, wherein the first purification treatment conditions are as follows:

a chromatographic column: WelFlash C18-I (20-40um,330 g); mobile phase: a is 0.05% formic acid solution, B is 0.05% methanol; flow rate of mobile phase: 90 ml/min; detection wavelength: 210nm &254 nm; controlling the pressure to be 90-150 psi; room temperature operation (20-25 deg.C);

gradient elution procedure: 0-16.8 min: a 80% → 69.8%; 16.8-17 min: a 69.8% → 0%; 17-23 min: a0%;

the amount of the hydrolysis reaction liquid injected into the medium pressure hplc preparative phase chromatograph in the present embodiment is applicable to other injection amounts such as 100-.

2. Second purification treatment

Concentrating (rotary evaporation concentrating) the first purified solution (10-11L) after the first purification treatment (i.e. medium pressure purification) until the first purified solution is concentrated into solid, and dissolving the solid into a first purified concentrated solution (50ml) by using a first diluent;

dividing the first purified concentrated solution into 10 parts, injecting sample for 10 times, each part being 5ml, injecting into a high pressure high performance liquid preparative chromatograph (model number BRIX 1860), performing second purification treatment (namely high pressure purification treatment), and collecting eluents with retention time of 16.8-18min and 19-20min respectively to obtain first and second characteristic peak purified solutions respectively; about 200-300ml of the first and second characteristic peak purified liquid is collected each time, the first and second characteristic peak eluents of the 10 times of second purification treatment are respectively combined, and the first and second characteristic peak purified liquid are about 2-3L respectively for standby, wherein the conditions of the second purification treatment are as follows:

a chromatographic column: ultimate XB-C18(250 x 50mm 10 μm); mobile phase: a is 0.05% formic acid solution, B is methanol; flow rate of mobile phase: 100 ml/min; first diluent: methanol: ultrapure water 1: 1; detection wavelength: 210nm &254 nm; controlling the pressure to be 2.5-3.4 MPa; room temperature operation (20-25 deg.C);

gradient elution procedure: 0-22 min: a 80% → 77%; 22-22.5 min: a 77% → 0%; 22.5-27 min: a is 0 percent;

the amount of the first purified concentrated solution injected into the hplc in this embodiment is also applicable to 5 ml/time, for example, 5 to 10 ml/time, and the amount of the reaction solution injected into the hplc during the second purification process is determined according to the size of the hplc.

3. Third purification treatment

Concentrating the purified solution (2-3L) with the first characteristic peak after the second purification treatment (i.e. rotary evaporation concentration), concentrating to solid, and dissolving with first diluent to obtain a first characteristic concentrated solution (50 ml);

dividing the first characteristic concentrated solution into 10 parts, injecting sample for 10 times, each part is 5ml, injecting into a high pressure high performance liquid preparative chromatograph (model number is BRIX 1860), performing third purification treatment (namely high pressure purification treatment), and collecting eluate with retention time of 26-28min to obtain third purified solution; about 200-300ml of the third purification solution is collected each time, the eluents of 10 times of the third purification treatment are combined, and the third purification solution is about 2-3L for standby, wherein the conditions of the third purification treatment are as follows:

a chromatographic column: ultimate XB-C18(250 x 50mm 7 μm); mobile phase: a is ultrapure water, and B is methanol; flow rate of mobile phase: 100 ml/min; first diluent: methanol: ultrapure water 1: 1; detection wavelength: 210nm &254 nm; controlling the pressure to be 2.5-3.4 MPa; room temperature operation (20-25 deg.C);

gradient elution procedure: 0-30 min: a 95% → 70%; 30-30.5 min: a 70% → 0%; 30.5-35 min: a is 0 percent;

4. fourth purification treatment

Concentrating (rotary evaporation concentrating) the second purified second characteristic peak purified solution (2-3L), concentrating to obtain solid, and dissolving with first diluent to obtain second characteristic concentrated solution (50 ml);

dividing the first characteristic concentrated solution into 10 parts, injecting sample for 10 times, each part is 5ml, injecting into a high pressure high performance liquid preparative chromatograph (model number is BRIX 1860), performing fourth purification treatment (namely high pressure purification treatment), and collecting eluate with retention time of 22-24min to obtain fourth purified solution; about 200-300ml of the fourth purification solution is collected each time, the eluents of 10 times of fourth purification treatment are combined, and the fourth purification solution is about 2-3L for standby, wherein the conditions of the fourth purification treatment are as follows:

a chromatographic column: ultimate XB-C18(250 x 50mm 7 μm); mobile phase: a is ultrapure water, and B is methanol; flow rate of mobile phase: 100 ml/min; first diluent: methanol: ultrapure water 1: 1; detection wavelength: 210nm &254 nm; controlling the pressure to be 2.5-3.4 MPa; room temperature operation (20-25 deg.C);

gradient elution procedure: 0-24 min: a 90% → 70%; 24-24.5 min: a 70% → 0%; 24.5-28 min: a is 0 percent;

5. identification of hydrolysate

Respectively detecting the purities of the third and fourth purified solutions with ultraviolet detector, wherein the purities of the substances in the third and fourth purified solutions are 99.41% and 98.66%, respectively, and the ultraviolet detection result is shown in FIG. 2; and then respectively carrying out freeze-drying treatment on the third purified liquid and the third purified liquid to respectively obtain a solid sample hydrolysate A and a solid sample hydrolysate B.

The hydrolysate A is white solid and is easily soluble in water; QTOF/MS mass spectrometry: parent ion (m/z-390.2500), daughter ion (m/z-372.2100, m/z-175.1300); the molecular formula is as follows: c₁₉H₁₉NO₈(ii) a Molecular weight: 390.1189, respectively; hydrolysate A was defined as oxytetracycline derivative M390-1.

The hydrolysate B is white solid and is easily dissolved in acetonitrile or methanol; QTOF/MS mass spectrometry: parent ion (m/z-390.2600), daughter ion (m/z-372.2000, m/z-175.1100); the molecular formula is as follows: c₁₉H₁₉NO₈(ii) a Molecular weight: 390.1189, respectively; hydrolysate B was defined as oxytetracycline derivative M390-2.

Nuclear Magnetic Resonance (NMR) analysis was performed on M390-1 and M390-2 to determine the C spectrum and H spectrum; the measurement results are shown in tables 1 and 2; the H spectrum and C spectrum of M390-1 are shown in FIGS. 3A and 3B; the H spectrum and C spectrum of M390-2 are shown in FIGS. 3C and 3D.

TABLE 1C-and H-spectra data of M390-1

TABLE 2C and H spectra data of M390-2

The structural formulas of M390-1 and M390-2 are respectively shown as formulas (I and II).

The M390-1 and M390-2 prepared by the invention can be used as standard substances for quantitatively detecting two hydrolysis products generated in the oxytetracycline hydrolysis process and other processes.

Example 3 detection of oxytetracycline derivative concentration

1. Preparing standard solution

Respectively and precisely weighing 10mg of each standard substance of the hydrolysate (M390-1 and M390-2) separated and purified in the example 2 into a brown small bottle, adding 10mL of methanol for dissolving, and respectively preparing M390-1 and M390-2 standard substance stock solutions with the concentration of 1000 mg/L;

respectively diluting the stock solutions (1000 mg/L) of the M390-1 and M390-2 standard products with ultrapure water to prepare 100mg/L standard solution; then, the M390-1 and M390-2 standard solutions are respectively diluted in a gradient way to prepare solutions for standard curves with the concentrations of 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01 for later use.

For example: taking 0.1mL of 1000mg/L stock solution into a 1.5mL brown sample bottle, adding 0.9mL of ultrapure water, diluting to 100mg/L standard solution, sequentially carrying out gradient dilution, and respectively diluting to obtain solutions for M390-1 and M390-2 standard curves with the concentrations of 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01.

2. Drawing a standard curve

Precisely absorbing 10 mul of each of the M390-1 and M390-2 standard curve solutions, respectively, injecting into a high performance liquid chromatography mass spectrometer (UPLC-MS/MS), and performing liquid chromatography mass spectrometry, wherein:

2A, chromatographic conditions are as follows:

a chromatographic column: waters ACUITY UPLC BEH (1.7 μm, 2.1 mm. times.100 mm); mobile phase: a is 0.1% formic acid solution, B is methanol; flow rate of mobile phase: 0.3 ml/min; column temperature: 35 ℃; sample temperature: 10 ℃; sample introduction amount: 10 mu L of the solution; gradient elution procedure: 0-1 min: a95%; 1-4.5 min: a 95% → 75%; 4.5-7 min: a 75% → 10%; 7-7.5 min: a 10% → 95%; 7.5-8.5 min: a95%;

2B, mass spectrometry conditions were as follows:

an ion source: electrospray ion source (ESI); the scanning mode is as follows: a positive ion mode; and (3) monitoring mode: multiple reaction detection mode (MRM); capillary voltage: 2.5 kV; taper hole voltage: 30V; source temperature: 150 ℃; desolventizing gas temperature: 500 ℃; desolventizing agent gas flow: 600L/h; taper hole gas flow: 60L/h;

respectively recording chromatographic peaks of M390-1 and M390-2, automatically drawing standard curves (as shown in FIGS. 4A and 4B) by a chromatographic workstation by taking peak areas as ordinate and sample concentration as abscissa, and respectively calculating a linear regression equation: the linear regression equation of M390-1 is that Y is 19.854x-535.49, R²0.9944, N8, showing that the linear relation of the concentration of the M390-1 sample is good between 10.00 and 1000.00 mu g/ml; the linear regression equation of M390-2 is that Y is 55.731x-1250.8, R²The linear relation between the concentration of the M390-2 sample and the concentration of the M390-2 sample is good when the concentration is 0.9975 and N is 8.

3. Results of chromatography and mass spectrometry

The chromatogram determination patterns of M390-1 and M390-2 are shown in FIG. 3, wherein the retention time of M390-1 is 6.26 min; the retention time of M390-2 is 6.49 min;

m390-1: parent ion (m/z-390.2500), daughter ion (m/z-372.2100, m/z-175.1300)

M390-2: parent ion (m/z-390.2600), daughter ion (m/z-372.2000, m/z-175.1100)

The chromatographic mass spectrometry conditions are suitable for quantitatively detecting M390-1 and M390-2 in the OTC hydrolysis process or hydrolysate, the chromatogram is shown in figure 3, wherein M390-1 is used for retention time of 6.26min, and M390-2 is used for retention time of 6.49min, and the two hydrolysates generated in the oxytetracycline hydrolysis process and other processes can be quantitatively detected by using the pure hydrolysate substances (M390-1 and M390-2) as standard substances.

Experimental example 1 sampling analysis and detection of oxytetracycline wastewater treatment full-flow water sample of oxytetracycline production factory (HS factory for short) in Shijiazhuang City of Hebei province

1. Waste water sampling

The process flow of the oxytetracycline waste water of the HS plant is shown in FIG. 4, wherein HS-ML is mother liquor, and the others are sampling points in the process respectively. The oxytetracycline mother liquor is waste liquor containing high-concentration oxytetracycline obtained by fermenting, separating and extracting a main product when an oxytetracycline HS manufacturer produces and manufactures the oxytetracycline, and the concentration of the oxytetracycline in the oxytetracycline mother liquor of the HS manufacturer is 451mg/L (usually 400 mg/L and 500 mg/L); in the wastewater treatment process, the water inflow of the oxytetracycline mother liquor is 539-794 m³D, 7 UASB reactors (volume 3100 m)³) The volume of the A/O pool is 5200m³。

Sampling from the hydrolysis treatment process of terramycin wastewater in an HS plant, wherein water samples are respectively marked as HS-ML, HS-1, HS-2, HS-3, HS-4 and HS-5, wherein:

HS-ML is obtained from oxytetracycline mother liquor, HS-1 is taken from wastewater after the oxytetracycline mother liquor is subjected to intensified hydrolysis treatment, HS-2 is taken from a distribution tank (about 3 times dilution), HS-3 is taken from a UASB anaerobic reactor, HS-4 is taken from an A/O aerobic reactor, and HS-5 is taken from wastewater in an aerobic sedimentation tank, as shown in figure 4. Each sample point was sampled 3 times, 3L each time.

2. Preparing standard solution

Preparing standard solutions of oxytetracycline derivatives M390-1, M390-2 at a concentration of 100mg/L according to the same method as instep 1 of example 3; then respectively carrying out gradient dilution to respectively dilute the solution into M390-1 and M390-2 standard curve solutions with the concentrations of 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01 for later use;

3. drawing M390-1 and M390-2 standard curves

According to the same method and chromatographic and mass spectrometric conditions as described instep 2 of example 3, M390-1 and M390-2 standard curves are respectively drawn, the chromatographic peaks of M390-1 and M390-2 are respectively recorded, the peak areas are used as ordinate, the sample concentration is used as abscissa, the standard curves are automatically drawn by the chromatographic workstation (as shown in FIGS. 4A and 4B), and the linear regression equation is respectively calculated: the linear regression equation of M390-1 is that Y is 19.854x-535.49, R²0.9944, N8, showing that the linear relation of the concentration of the M390-1 sample is good between 10.00 and 1000.00 mu g/ml; the linear regression equation of M390-2 is that Y is 55.731x-1250.8, R²The linear relation between the concentration of the M390-2 sample and the concentration of the M390-2 sample is good when the concentration is 0.9975 and N is 8.

4. Pretreatment of oxytetracycline wastewater

Because a large amount of matrix substances exist in the actual wastewater, the detection result is affected, and therefore, the actual water sample needs to be pretreated. The pretreatment steps are as follows:

4-1) solid phase extraction:

activating a solid phase extraction column: taking a solid phase extraction column (Oasis HLB solid phase extraction column, 500mg, 6cc, Waters), and respectively activating the solid phase extraction column with 5mL of dichloromethane, 5mL of methanol and 5mL of phosphate buffer solution (pH is 3.0);

sample preparation: respectively filtering wastewater samples (HS-ML, HS-1, HS-2, HS-3, HS-4 and HS-5) collected from a full-flow process collection point in the process of treating the oxytetracycline wastewater of an HS plant by using a 0.45 mu m aqueous phase filter membrane, respectively adding 50mL of the filtered wastewater samples into 500mL of ultrapure water, respectively adding Na into the ultrapure water₂EDTA (0.1g, usually 0.05-0.2 per 50ml filtrate) as metal chelating agent to remove interference of metal ions in the wastewater, adjusting pH to 3 with dilute hydrochloric acid or sodium hydroxide solution, and performing solid phase extraction;

enriching a water sample: loading the sample at the speed of about 10mL/min, enriching the sample into a solid phase extraction cartridge, washing the filter cartridge with 6mL of ultrapure water after the water sample is passed, drying the loaded solid phase extraction cartridge with nitrogen, and storing at-20 ℃ for later use. The purpose of drying with nitrogen is to prevent the interference of oxygen and carbon dioxide contained in the air with the sample, and nitrogen does not cause oxidation.

4-2) preparing sample detection diluent

Eluting the solid phase extraction column by 5mL of methanol, concentrating the eluent under mild nitrogen flow to dryness, and dissolving the eluent in 0.5mL of methanol to obtain a solid phase extraction-concentrated sample solution;

prior to LC-MS/MS (liquid phase secondary mass spectrometry) analysis, the solid phase extracted-concentrated sample solution was diluted with pure water, wherein the volume ratio of the solid phase extracted-concentrated sample solution to the pure water was 1: 1(V/V), namely adding 0.5ml of pure water, and diluting into a detection diluent (1 ml);

5. LC-MS/MS analysis and detection of terramycin wastewater

Performing LC-MSMS analysis detection on the detection diluent, wherein:

5A, the chromatographic conditions for analysis and detection are as follows:

a chromatographic column: waters ACUITY UPLC BEH (1.7 μm, 2.1 mm. times.100 mm); mobile phase: a is 0.1% formic acid solution, B is methanol; flow rate of mobile phase: 0.3 ml/min; column temperature: 35 ℃; sample temperature: 10 ℃; sample introduction amount: 10 mu L of the solution;

gradient elution procedure: 0-1 min: a95%; 1-4.5 min: a 95% → 75%; 4.5-7 min: a 75% → 10%; 7-7.5 min: a 10% → 95%; 7.5-8.5 min: a95%;

5B, analyzing and detecting mass spectrum conditions as follows:

an ion source: electrospray ion source (ESI); the scanning mode is as follows: a positive ion mode; and (3) monitoring mode: multiple reaction detection mode (MRM); capillary voltage: 2.5 kV; taper hole voltage: 30V; source temperature: 150 ℃; desolventizing gas temperature: 500 ℃; desolventizing agent gas flow: 600L/h; taper hole gas flow: 60L/h.

6. The result of the detection

Chromatograms of M390-1 and M390-2 substances detected from HS actual wastewater and their standards are shown in FIG. 5, and retention times of the two substances detected in the actual wastewater respectively correspond to retention times of M390-1 and M390-2 standard substances according to the retention time comparison, thereby confirming that they are M390-1 and M390-2.

After confirming that the substances are corresponding M390-1 and M390-2, the concentrations of the two hydrolysates in the samples collected from the corresponding treatment steps of the actual wastewater are calculated according to the peak area-concentration corresponding relationship according to the standard curves of M390-1 and M390-2 drawn instep 3, as shown in FIG. 6.

It should be noted that both of the HS-1 to HS-2 hydrolysates in FIG. 6 were greatly reduced because of the dilution effect of about 3 times that caused by the addition of other sewage after hydrolysis.

The detection result shows that after the actual oxytetracycline production mother liquor is subjected to intensified hydrolysis, the sum of the concentrations of M390-1 and M390-2 generated in an HS-1 sample reaches 80mg/L, wherein the concentration of M390-1 is 50mg/L, and the concentration of M390-2 is 30 mg/L; after dilution in the distribution tank, the concentrations of M390-1 and M390-2 were reduced to 21mg/L and 13mg/L, respectively. The concentration of M390-1 in the HS-3 sample treated by the UASB reactor, the HS-4 sample treated by the A/O reactor and the HS-5 sample treated by the aerobic sedimentation tank is respectively as follows: 18mg/L, 15mg/L, 14 mg/L; the concentration of M390-2 is respectively as follows: 10mg/L, 8mg/L, 7 mg/L. From the measurement results, it was found that: the biological treatment of the waste water after the terramycin hydrolysis treatment can not well remove terramycin hydrolysis products M390-1 and M390-2.

The detection of the corresponding concentrations of the two substances M390-1 and M390-2 in the whole wastewater treatment process indicates that the two substances cannot be well removed by the conventional wastewater treatment method, and particularly, the two substances still have higher concentrations after biological treatment, which may cause the consequence that the COD in the wastewater is difficult to effectively remove.

Test example 2: sampling, analyzing and detecting a water sample of a small oxytetracycline waste water treatment device of a certain oxytetracycline manufacturer (SX factory for short) in Shijiazhuang city in Hebei province

1. Waste water sampling

The wastewater treatment flow of the small test device of the SX plant is shown in FIG. 7, wherein SX-ML is mother liquor, and the others are sampling points in the process. The oxytetracycline mother liquor is waste liquor containing high-concentration oxytetracycline obtained by fermenting, separating and extracting a main product when an oxytetracycline SX factory produces the oxytetracycline, and the concentration of the oxytetracycline in the oxytetracycline mother liquor of an XS factory is 495mg/L (usually 400-500 mg/L); in the wastewater treatment process, the water inflow of the oxytetracycline mother liquor is 10L/d, the water amounts of the two UASB reactors are 960mL/d and 2060mL/d respectively, the water amount of the aerobic reactor is 420mL/d, and the effective volumes of the reactors are 3L.

Sampling from the oxytetracycline wastewater hydrolysis treatment process of an SX factory, wherein water samples are marked as SX-ML, SX-1, SX-2, SX-3, SX-4 and SX-5 respectively, wherein:

SX-ML is obtained from oxytetracycline mother liquor, SX-1 is taken from wastewater after the oxytetracycline mother liquor is subjected to intensified hydrolysis treatment in a small test device, SX-2 is taken from a water sample after hydrolysis and coagulation post-treatment, SX-3 is taken from a UASB anaerobic reactor No. 1, SX-4 is taken from a UASB anaerobic reactor No. 2, SX-5 is taken from the UASB reactors No. 1 and No. 2 according to the weight ratio of 1:1, taking the SX-6 water sample from the wastewater in the aerobic reactor. Each sample point was sampled 3 times, 3L each time.

2. Preparing standard solution

Preparing standard solutions of oxytetracycline derivatives M390-1, M390-2 at a concentration of 100mg/L according to the same method as instep 1 of example 3; then respectively carrying out gradient dilution to respectively dilute the solution into M390-1 and M390-2 standard curve solutions with the concentrations of 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01 for later use;

3. drawing M390-1 and M390-2 standard curves

According to the same method and chromatographic and mass spectrometric conditions as instep 2 of example 3, M390-1 and M390-2 standard curves are respectively drawn, chromatographic peaks of M390-1 and M390-2 are respectively recorded, standard curves (as shown in FIGS. 4A and 4B) are automatically drawn by a chromatographic workstation with peak areas as ordinate and sample concentration as abscissa, and linear regression equations are respectively calculated: the linear regression equation of M390-1 is that Y is 19.854x-535.49, R²0.9944, N8, showing that the linear relation of the concentration of the M390-1 sample is good between 10.00 and 1000.00 mu g/ml; the linear regression equation of M390-2 is that Y is 55.731x-1250.8, R²＝0.9975，N＝8，The linear relation of the concentration of the M390-2 sample between 10.00 and 1000.00 mu g/ml is good.

4. Pretreatment of oxytetracycline wastewater

Because a large amount of matrix substances exist in the actual wastewater, the detection result is affected, and therefore, the actual water sample needs to be pretreated. The pretreatment steps are as follows:

4-1) solid phase extraction:

activating a solid phase extraction column: taking a solid phase extraction column (Oasis HLB solid phase extraction column, 500mg, 6cc, Waters), and respectively activating the solid phase extraction column with 5mL of dichloromethane, 5mL of methanol and 5mL of phosphate buffer solution (pH is 3.0);

sample preparation: filtering wastewater samples (SX-ML, SX-1, SX-2, SX-3, SX-4 and SX-5) collected from a full-flow process collection point in the process of treating the oxytetracycline wastewater from an SX plant by adopting a 0.45-micron aqueous phase filter membrane, taking 50mL of each filtered wastewater sample, respectively adding ultrapure water to 500mL, adding Na, and filtering₂EDTA (0.1g, usually 0.05-0.2g) as metal chelating agent to remove interference of metal ions in the wastewater, adjusting pH to 3 with dilute hydrochloric acid or sodium hydroxide solution, and performing solid phase extraction;

enriching a water sample: loading the sample at the speed of about 10mL/min, enriching the sample into a solid phase extraction cartridge, washing the filter cartridge with 6mL of ultrapure water after the water sample is passed, drying the loaded solid phase extraction cartridge with nitrogen, and storing at-20 ℃ for later use.

4-2) preparing sample detection diluent

Eluting the solid phase extraction column by 5mL of methanol, concentrating the eluent under mild nitrogen flow to dryness, and dissolving the eluent in 0.5mL of methanol to obtain a solid phase extraction-concentrated sample solution;

or, eluting the solid phase extraction column with 5mL of methanol, and concentrating the eluent to 0.5mL under mild nitrogen flow to obtain a solid phase extraction-concentrated sample solution;

prior to LC-MSMS analysis, the solid phase extraction-concentrated sample solution was purified water at a ratio of 1: diluting at a ratio of 1(V/V), namely adding 0.5ml of pure water, and diluting into a detection diluent (1 ml);

5. LC-MSMS analysis and detection of terramycin wastewater

The test dilution was subjected to LC-MSMS analysis and detection under the same chromatographic conditions and mass spectrometric conditions as those in test example 1.

6. The result of the detection

The chromatograms of M390-1 and M390-2 substances detected in SX actual wastewater and their standards are shown in FIG. 8, and the retention times of the two substances detected in the actual wastewater correspond to the retention times of the M390-1 and M390-2 standard substances, respectively, according to the comparison of the retention times, thereby confirming that they are M390-1 and M390-2.

After confirming that the substances are corresponding M390-1 and M390-2, the concentrations of the two hydrolysates in the samples collected from the corresponding treatment steps of the actual wastewater were calculated from the peak area-concentration correspondence according to the standard curves of M390-1 and M390-2 measured instep 3, as shown in FIG. 9.

The detection result shows that after the actual oxytetracycline production mother liquor of an SX plant is subjected to intensified hydrolysis, the sum of the concentrations of M390-1 and M390-2 generated in an SX-1 sample reaches 120mg/L, wherein the concentration of M390-1 is 69mg/L, and the concentration of M390-2 is 51 mg/L. After coagulation, the concentrations of M390-1 and M390-2 were not reduced and remained at 69mg/L and 51 mg/L. After the treatment of the UASB reactor No. 1 (SX-3), the concentrations of M390-1 and M390-2 are respectively 61mg/L and 47 mg/L; after the treatment of the UASB reactor No. 2 (SX-4), the concentrations of M390-1 and M390-2 are 59mg/L and 47mg/L respectively; after water samples of the two anaerobic reactors are mixed, the concentrations of M390-1 and M390-2 are respectively 58mg/L and 45 mg/L; after passing through the aerobic reactor, the concentrations of M390-1 and M390-2 in the effluent are respectively 56mg/L and 44 mg/L.

It can be seen that the concentrations of M390-1 and M390-2 did not decrease throughout the entire process of the SX pilot plant, and both materials were detected at corresponding concentrations throughout the wastewater treatment process, indicating that both materials could not be removed well by conventional wastewater treatment methods, especially at higher concentrations after biological treatment, which may have the consequence that the COD of the wastewater was difficult to remove effectively.

Because M390-1 and M390-2 with higher concentration are detected in the whole actual wastewater treatment process, which indicates that the biodegradability is poor, and higher concentration is still detected in biological effluent, the COD component which is difficult to biodegrade can be formed, and the toxicity, the titer and other properties of the COD component can cause potential environmental risks, therefore, effective removal methods of the two substances are still to be researched to effectively control the COD component and the COD component, so that increasingly strict environmental quality control standards are met.

The above-described embodiments of the present invention are merely exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (10)

Translated fromChinese

1.一种如结构式(Ⅰ)所述的土霉素衍生物M390-1：1. An oxytetracycline derivative M390-1 as described in structural formula (I):

2.一种如结构式(Ⅱ)所述的土霉素衍生物M390-2：2. An oxytetracycline derivative M390-2 as described in structural formula (II):

3.一种制备土霉素衍生物M390-1、M390-2的方法，其特征是，包括首先对土霉素进行水解处理，制备土霉素水解溶液；然后对土霉素水解溶液采用高效液相色谱法进行分离、纯化处理。3. a method for preparing oxytetracycline derivatives M390-1, M390-2, is characterized in that, comprises at first hydrolyzing oxytetracycline to prepare oxytetracycline hydrolyzed solution; Separation and purification by liquid chromatography.4.一种制备土霉素衍生物M390-1、M390-2的方法，其特征是，包括如下进行的步骤：4. a method for preparing oxytetracycline derivatives M390-1, M390-2, is characterized in that, comprises the step of carrying out as follows:1)将土霉素水解溶液或土霉素生产废水注入中压高效液相制备色谱仪中，进行第一纯化处理(即中压纯化处理)，根据保留时间收集洗脱液，获得第一纯化液；1) inject the oxytetracycline hydrolyzed solution or the oxytetracycline production wastewater into the medium pressure high performance liquid preparative chromatograph, carry out the first purification treatment (that is, the medium pressure purification treatment), collect the eluent according to the retention time, and obtain the first purification liquid;2)对第一纯化液浓缩后，注入高压高效液相制备色谱仪中进行第二纯化处理，根据保留时间进行洗脱液收集，分别获得第一、第二特征峰纯化液；2) after concentrating the first purified solution, inject it into a high pressure high performance liquid phase preparative chromatograph to carry out the second purification process, and collect the eluent according to the retention time to obtain the first and second characteristic peak purified solutions respectively;3)将第一特征峰纯化液浓缩后，注入高压高效液相制备色谱仪中进行第三纯化处理，根据保留时间收集洗脱液，获得土霉素衍生物M390-1；3) after concentrating the first characteristic peak purified solution, inject it into a high pressure high performance liquid preparative chromatograph for the third purification process, collect the eluent according to the retention time, and obtain the oxytetracycline derivative M390-1;4)将第二特征峰纯化液浓缩后，注入高压高效液相制备色谱仪中进行第四纯化处理，根据保留时间收集洗脱液，获得土霉素衍生物M390-2。4) After concentrating the purified liquid of the second characteristic peak, inject it into a high pressure high performance liquid preparative chromatograph to carry out the fourth purification treatment, collect the eluate according to the retention time, and obtain the oxytetracycline derivative M390-2.5.如权利要求4所述的方法，其特征是，步骤1)中收集保留时间为13-15.8min的洗脱液，获得所述第一纯化液；步骤2)中收集保留时间为16.8-18min的洗脱液，获得所述第一特征峰纯化液；收集保留时间为19-20min的洗脱液，获得所述第二特征峰纯化液；步骤3)中收集保留时间为26-28min的洗脱液，并浓缩、干燥，获得所述的土霉素衍生物M390-1；步骤4)中收集保留时间为22-24min的洗脱液，并浓缩、干燥，获得所述的土霉素衍生物M390-2。5. method as claimed in claim 4, is characterized in that, collecting retention time in step 1) is the eluent of 13-15.8min, obtains described first purified solution; In step 2), collecting retention time is 16.8- 18min of eluent to obtain the first characteristic peak purified solution; to collect the eluent with a retention time of 19-20min to obtain the second characteristic peak purified solution; in step 3), to collect the eluent with a retention time of 26-28min The eluate was concentrated and dried to obtain the oxytetracycline derivative M390-1; in step 4), the eluate with a retention time of 22-24 min was collected, concentrated and dried to obtain the oxytetracycline derivative Derivative M390-2.6.如权利要求4或5所述的方法，其特征是，步骤1)中所述第一纯化处理的流动相为：A为0.05％的甲酸溶液，B为0.05％的甲醇；步骤2)中所述第二纯化处理的流动相为：A为0.05％的甲酸溶液，B为甲醇；步骤3)中所述第三纯化处理的流动相为：A为超纯水，B为甲醇；步骤4)中所述第四纯化处理的流动相为：A为超纯水，B为甲醇。6. The method according to claim 4 or 5, wherein the mobile phase of the first purification treatment in step 1) is: A is 0.05% formic acid solution, B is 0.05% methanol; step 2) The mobile phase of the second purification treatment described in the above is: A is 0.05% formic acid solution, and B is methanol; the mobile phase of the third purification treatment described in step 3) is: A is ultrapure water, and B is methanol; step The mobile phase of the fourth purification treatment described in 4) is: A is ultrapure water, and B is methanol.7.一种测定土霉素废水中土霉素衍生物M390-1、M390-2含量的方法，其特征是，包括如下步骤：7. a method for measuring the content of oxytetracycline derivatives M390-1 and M390-2 in oxytetracycline waste water, is characterized in that, comprises the steps:A)用土霉素衍生物M390-1、390-2标准品分别配制浓度为1、0.75、0.5、0.25、0.1、0.05、0.025、0.01mg/L的M390-1、390-2标准曲线用溶液；A) Use oxytetracycline derivatives M390-1 and 390-2 standard products to prepare M390-1 and 390-2 standard curve solutions with concentrations of 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01 mg/L respectively ;B)分别精密吸取M390-1和M390-2标准曲线用溶液，分别注入高效液相色谱仪中，进行液相色谱测定，分别记录并测定步骤A)中所述浓度下，土霉素衍生物M390-1、M390-2的色谱峰、保留时间、峰面积；B) Accurately draw the solutions for M390-1 and M390-2 standard curve respectively, inject them into the high performance liquid chromatograph respectively, carry out liquid chromatographic determination, respectively record and measure the concentration described in step A), the oxytetracycline derivative Chromatographic peaks, retention times and peak areas of M390-1 and M390-2;C)分别以土霉素衍生物M390-1、M390-2的色谱峰的峰面积为纵坐标、以浓度为横坐标，分别绘制M390-1、M390-2的标准曲线，其中：C) Take the peak area of the chromatographic peaks of the oxytetracycline derivatives M390-1 and M390-2 as the ordinate and the concentration as the abscissa, respectively draw the standard curves of M390-1 and M390-2, wherein:土霉素衍生物M390-1的线性回归方程为：Y1＝19.854X1-535.49；其中，Y1为M390-1的峰面积，X1为M390-1的浓度；The linear regression equation of oxytetracycline derivative M390-1 is: Y1=19.854X1-535.49; wherein, Y1 is the peak area of M390-1, and X1 is the concentration of M390-1;土霉素衍生物M390-2的线性回归方程为：Y2＝55.731X2-1250.8，其中，Y2为M390-2的峰面积，X2为M390-2的浓度；The linear regression equation of oxytetracycline derivative M390-2 is: Y2=55.731X2-1250.8, wherein, Y2 is the peak area of M390-2, and X2 is the concentration of M390-2;D)土霉素废水预处理后制成检测稀释液，然后进行LC-MS/MS分析检测，分别与标准品M390-1、M390-2的保留时间相比对，分别记录、测定与标准品M390-1、M390-2的保留时间相对应的土霉素废水中M390-1、M390-2的色谱峰及峰面积；D) After pretreatment of oxytetracycline wastewater, a detection diluent is prepared, and then LC-MS/MS analysis and detection are carried out. The chromatographic peaks and peak areas of M390-1 and M390-2 in the oxytetracycline wastewater corresponding to the retention times of M390-1 and M390-2;E)将步骤D)测定的M390-1、M390-2的峰面积分别代入对应的线性回归方程，计算得到M390-1、M390-2的浓度。E) Substitute the peak areas of M390-1 and M390-2 determined in step D) into the corresponding linear regression equations respectively, and calculate the concentrations of M390-1 and M390-2.8.如权利要求7所述的方法，其特征是，步骤B)中所述液相色谱测定的色谱条件如下：流动相：A为0.1％的甲酸溶液，B为甲醇；梯度洗脱程序：0-1min：A 95％；1-4.5min：A 95％→75％；4.5-7min：A 75％→10％；7-7.5min：A 10％→95％；7.5-8.5min：A 95％。8. method as claimed in claim 7 is characterized in that, the chromatographic condition of liquid chromatography described in step B) is as follows: mobile phase: A is 0.1% formic acid solution, and B is methanol; Gradient elution program: 0-1min: A 95%; 1-4.5min: A 95%→75%; 4.5-7min: A 75%→10%; 7-7.5min: A 10%→95%; 7.5-8.5min: A 95 %.9.如权利要求7或8所述的方法，其特征是，步骤D)中所述检测稀释液按照如下步骤制成：D1)将土霉素废水采用0.45μm水相滤膜过滤，向滤液中加入超纯水、金属螯合剂，并用盐酸或氢氧化钠溶液调整pH至3，获得过滤样品液；9. method as claimed in claim 7 or 8, is characterized in that, described in step D), detects diluent and is made according to the following steps: D1) oxytetracycline waste water is filtered by 0.45 μm water-phase membrane filter, to filtrate Add ultrapure water and metal chelating agent, and adjust pH to 3 with hydrochloric acid or sodium hydroxide solution to obtain filtered sample solution;D2)对过滤样品液进行固相萃取处理，固相萃取过程中采用甲醇洗脱，洗脱液干燥后，用甲醇溶解，获得固相萃取-浓缩样品液；然后加入超纯水，稀释成检测稀释液。D2) Perform solid-phase extraction treatment on the filtered sample solution. During the solid-phase extraction process, methanol is used for elution. After the eluent is dried, it is dissolved in methanol to obtain a solid-phase extraction-concentrated sample solution; then ultrapure water is added to dilute to detect Diluent.10.如权利要求7或8所述的方法，其特征是，步骤D)所述LC-MS/MS分析检测过程中，色谱条件如下：流动相：A为0.1％的甲酸溶液，B为甲醇；梯度洗脱；质谱条件如下：离子源：电喷雾离子源ESI；扫描方式：正离子方式。10. the method as claimed in claim 7 or 8 is characterized in that, in step D) in the described LC-MS/MS analysis detection process, chromatographic condition is as follows: mobile phase: A is 0.1% formic acid solution, and B is methanol ; gradient elution; mass spectrometry conditions are as follows: ion source: electrospray ion source ESI; scanning mode: positive ion mode.

Images