Impurity detection method of terlipressin preparation for injection

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an impurity detection method of a terlipressin preparation for injection. The impurity is the deamination esterification impurity of terlipressin and mannitol found for the first time, the structure of the impurity is shown as a formula I and / or a formula II, and the synthesized impurity is used as a reference substance and is detected through ultra-high performance liquid chromatography.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for detecting impurities in injectable terlipressin preparations. Background Technology

[0002] Terlipressin is a chemically synthesized polypeptide drug composed of 12 amino acids condensed together. Its chemical name is glycine-glycine-glycine-L-cysteyl-L-tyrosyl-L-phenylalanyl-L-glutamine-L-asparaginyl-L-cysteyl-L-prolyl-L-lysyl-glycineamine (4→9 disulfide ring), and its molecular formula is C1. 52 H 74 N 16 O 15 S2, CAS Registry No. 14636-12-5. Terlipressin is an analogue of a hormone secreted by the posterior pituitary gland. In vivo, it is slowly released as active lysine vasopressin after the removal of three glycine residues from its N-terminus by aminopeptidase, producing a contractile effect on smooth muscle. Clinically, it is mainly used to treat esophageal variceal bleeding. In some countries, it is approved for the treatment of type I hepatorenal syndrome and is the only first-line drug approved by the FDA for hepatorenal syndrome and renal failure. Terlipressin for Injection is developed by Ferring Pharmaceuticals GmbH in Germany and marketed as Glypressin. By 2025, nearly 20 generic drug companies in China will be producing this product. The dosage form is an injection, with a strength of 1 mg (equivalent to 0.86 mg terlipressin), and the main excipient is mannitol. Terlipressin can generate impurities during storage; identifying and detecting these impurities is beneficial for improving drug quality and safety.

[0003] Upgraded liquid chromatography (UPLC) is one of the most commonly used separation and analysis techniques in chemistry, medicine, and other fields, especially suitable for detecting trace impurities in pharmaceuticals. It uses a high-pressure pump to deliver the mobile phase and sample into a chromatographic column, achieving separation by utilizing the differences in the interactions between different components and the stationary phase, followed by analysis using a detector. Compared to traditional methods, UPLC offers strong separation capabilities, fast analysis speed, and effective control over pharmaceutical quality. CN115856149A discloses a method for detecting impurities using high-performance liquid chromatography, where terlipressin is diluted with the mobile phase as the sample to be detected, and the sample is diluted 100 times as a reference standard, using trifluoroacetic acid... water Acetonitrile is used as the mobile phase to obtain chromatograms of the sample to be tested and the control sample, thereby determining the content of impurities. CN103675138B discloses an ultra-high performance liquid chromatography (UHPLC) method for separating and analyzing terlipressin and its impurities. The method includes using mobile phases A and B, prepared with buffer and organic solvent in different proportions, as mobile phases in an UHPLC system to perform gradient elution of terlipressin and its impurities, thereby rapidly separating and analyzing terlipressin and its impurities.

[0004] During the research on the compatibility of raw materials and excipients, it was found that the main component of injectable terlipressin reacts directly with the non-reducing sugar excipient mannitol to generate a new compound impurity. It is necessary to conduct structural confirmation, preparation, quality characterization and application research on this impurity to provide a basis for the quality control of the formulation. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting impurities generated by the reaction of terlipressin and mannitol in formulations using ultra-high performance liquid chromatography.

[0006] The present invention adopts the following technical solution: A quality control detection method for injectable terlipressin preparations, employing ultra-high performance liquid chromatography, using impurities of formula I and / or formula II as impurity reference standards, to detect the content of the impurities, wherein the injectable terlipressin preparations contain terlipressin and mannitol; Formula I .

[0007] Formula I is the compound formed by the reaction of glycine at position 12 of terlipressin with mannitol after deamination, and Formula II is the compound formed by the deamination and esterification reaction of asparagine at position 8 of terlipressin with mannitol. Both formulas have a single isotopic molecular weight of 1391.5510. The injectable terlipressin formulation can be a conventional lyophilized powder, generally containing terlipressin, mannitol (excipient), pH adjuster, etc., for example, the mass ratio of terlipressin to mannitol can be 1:10.

[0008] Furthermore, the ultra-high performance liquid chromatography method includes the following steps: (1) Prepare impurity reference solution and test solution, wherein the test solution is terlipressin for injection; (2) In an ultra-high performance liquid chromatograph, a C18 column is used, and mobile phase A and mobile phase B are used to elute the impurity reference standard and the test sample at a flow rate of 0.1~0.5 ml / min. The detection wavelength is 200~400 nm to obtain a chromatogram and determine the impurity content.

[0009] Furthermore, the impurity content refers to the total content of esterified impurities of terlipressin and mannitol, including Formula I, Formula II, or other deamination-esterified impurities, such as the esterification of glutamine acyl at position 7 with mannitol. Since Formula I, Formula II, or other deamination-esterified impurities cannot be distinguished by high-performance liquid chromatography, the detection is based on the total content of esterified impurities of terlipressin and mannitol. The reference standard can be impurities of Formula I or Formula II, as well as mixtures of the two.

[0010] Furthermore, the mobile phase A is acetonitrile:water = 90:10 and contains 0.1% anhydrous formic acid, and the mobile phase B is acetonitrile:water = 60:40 and contains 0.1% anhydrous formic acid.

[0011] Furthermore, the gradient elution procedure is as follows: in the mobile phase, the initial gradient mobile phase B ratio is 0%, the mobile phase B ratio increases from 0% to 100% from 0 to 10 min, the mobile phase B ratio is 100% from 10 to 15 min, the mobile phase B ratio decreases from 100% to 0% from 15 to 16 min, and the mobile phase B ratio is 0% from 16 to 25 min.

[0012] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention is the first to discover and confirm the impurities generated by the reaction of the 12th and 8th amino acids of terlipressin with mannitol in injectable terlipressin formulations, and provides a high-performance liquid chromatography detection method that can detect the total content of esterified impurities of terlipressin and mannitol, not limited to impurities of Formula I or Formula II. Attached Figure Description

[0013] Figure 1 The image shown is a primary mass spectrum of mannitol esterification impurities.

[0014] Figure 2 The image shown is a mass spectrometry fragment matching diagram of the mannitol esterification impurities represented by Formula I.

[0015] Figure 3 The image shown is a mass spectrometry fragment matching diagram of the mannitol esterification impurities represented by Formula II.

[0016] Figure 4 The diagram shows the reaction mechanism of mannitol deamination to esterification and the formation of impurities.

[0017] Figure 5 The image shown is a secondary fragmentation diagram of the confirmatory mass spectrometry structure of the mannitol esterified impurity reference standard shown in Formula I.

[0018] Figure 6 The diagram shows the identification of the NMR chemical shift of the mannitol esterification reference structure represented by Formula I.

[0019] Figure 7The image shown is a secondary fragmentation mass spectrometry diagram confirming the structure of the mannitol esterified impurity reference standard shown in Formula II.

[0020] Figure 8 The diagram shows the identification of the NMR chemical shift of the mannitol esterification reference structure as shown in Formula II.

[0021] Figure 9 The image shows the ultra-high performance liquid chromatogram of the impurity represented by Formula I.

[0022] Figure 10 The image shows the ultra-high performance liquid chromatogram of the impurity represented by Formula II.

[0023] Figure 11 This is the ultra-high performance liquid chromatogram of the test sample. Detailed Implementation

[0024] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0025] The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, which should be understood to include those close to them.

[0026] Example 1: Discovery and analysis of impurities in the esterification process at position 12 (deamination) During a compatibility study of terlipressin with its raw materials and excipients, the inventors discovered an anomaly at the shoulder of the main peak. They collected the effluent and performed first-order mass spectrometry analysis. In addition to the main component (mass-to-charge ratio 614.2568), another signal with a mass-to-charge ratio of 696.7833 was observed in the mass spectrum. Figure 1 As shown in the figure. Further secondary mass spectrometry analysis yielded secondary fragment results for two similar impurities. The secondary fragment matching diagram for one impurity with a higher content is shown in the figure. Figure 2 As shown in Table 1, the mass spectrometry fragment matching diagrams for impurities with lower content are as follows: Figure 3 As shown in Table 2, the measured secondary fragments of the impurity with higher content match perfectly with the theoretical fragment of the +165.0530 modification at position 12, confirming that the terminal amide at position 12 underwent deamination and esterification. The measured secondary fragments of the impurity with lower content match perfectly with the theoretical fragment of the +165.0530 modification at position 8, confirming that the amide at position 8 underwent deamination and esterification. Therefore, the structural formulas of the mannitol deamination and esterification impurities are shown in Formulas I and II above, with a single isotopic molecular weight of 1391.5510.

[0027] The analysis was performed using a Thermo Vanquish UPLC-orbitrap exploris 480 high-resolution mass spectrometer.

[0028] Chromatographic conditions: Waters peptide BEH C18 column (2.1 mm × 100 mm, 1.7 μm), mobile phase A was 0.1% formic acid water (v:v) solution, mobile phase B was acetonitrile solution, gradient elution was performed, flow rate was 0.25 ml / min, column temperature was 35 ℃, injection volume was 2 μL, and detection wavelength was 210 nm.

[0029] Mass spectrometry conditions: Acquisition mode: DDA; Ion source: H-ESI; Positive ion mode; Mass spectrometer spray voltage: 3400V; MS scan range (m / z): 300-1800; Resolution: 120000; RF Lens (%): 50; Ion transfer tube: 320℃; Desolvation gas: 400℃; Sheath gas: 35Arb; Auxiliary gas: 10Arb; Purge gas: 0Arb; Fragmentation mode: HCD.

[0030] According to the inventor's analysis, the impurity formation mechanism is as follows: Figure 4 As shown: In this process, the 12-position amide undergoes carbonyl protonation under strong acidic conditions, which enhances its electrophilicity. The ortho-imide undergoes a keto-enol tautomerization, nucleophilically attacking the protonated carbonyl group for deamination. Through proton exchange, a stable oxo-oxo-nitrogen heterocyclic five-membered ring transition state is formed. Under strong acidic conditions, the carbonyl group undergoes carbonyl protonation, and the mannitol hydroxyl group nucleophilically attacks the protonated carbonyl group for ring opening, resulting in a low-energy transesterification reaction, which indirectly achieves deamination and esterification.

[0031] Under relatively strong acidic conditions, the carbonyl group of asparagine at the 8-position is protonated, which enhances its electrophilicity. The ortho-imide nucleophilically attacks the protonated carbonyl group to deamination, forming a transition state of the classic succinimide five-membered ring structure for peptide degradation through proton exchange. The two carbonyl groups need to share the lone pair electrons of the nitrogen atom, which further enhances the electrophilicity. Under relatively strong acidic conditions, the mannitol hydroxyl group also undergoes nucleophilic attack, which also achieves deamination and esterification.

[0032] Example 2: Preparation of 12-position deamination to esterification impurities (Formula I impurities) by solid-phase synthesis method There is a deamination and esterification reaction between terlipressin and the non-reducing sugar mannitol, which occurs at the 12th position of aminoglycine.

[0033] The preparation of the 12-position deamination and esterification impurity involves first synthesizing the terlipressin peptide chain stepwise on a solid-phase support, followed by reaction with mannitol, and includes the following steps: (1) Weigh 2-CTC resin and place it in a reaction tube, add DCM to swell it. After swelling, add DMF to wash it, then add Fmoc-Gly-OH (2 eq), DIEA (4 eq), and DCM to condense it for 90 min. After the reaction is complete, add methanol (5 eq) to cap the resin for 20 min, then add DMF to wash it.

[0034] (2) Add DBLK solution (20% pip / DMF solution) for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Lys(Boc)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and proceeded to the next condensation cycle.

[0035] (3) Add DBLK solution (20% pip / DMF solution) for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Pro-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and entered the next condensation cycle.

[0036] (4) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Cys(Trt)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. Take the resin and test it by Kaiser method. If the resin is colorless, proceed to the next condensation cycle.

[0037] (5) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Asn(Trt)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and entered the next condensation cycle.

[0038] (6) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Gln(Trt)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and entered the next condensation cycle.

[0039] (7) Add DBLK solution (20% pip / DMF solution) for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Phe-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin is tested by Kaiser method. If the resin is colorless, proceed to the next condensation cycle.

[0040] (8) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Tyr(tBu)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and entered the next condensation cycle.

[0041] (9) Add DBLK solution (20% pip / DMF solution) for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Cys(Trt)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. Take the resin and test it by Kaiser method. If the resin is colorless, proceed to the next condensation cycle.

[0042] (10) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Gly-Gly-Gly-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. Take the resin and test it by Kaiser method. If the resin is colorless, add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. Then add ether for washing and dry the resin.

[0043] (11) Add the peptide resin to the 20% TFE / DCM (m / V=1:10) cleavage solution and react for 90 min. After the reaction is completed, dry by rotary evaporation to obtain the fully protected peptide.

[0044] (12) Dissolve the fully protected peptide in DCM, add mannitol, HOBT and DIC (2 eq each) and condense for 90-150 min. After the reaction is complete, remove the liquid by rotary evaporation to obtain the solid.

[0045] (13) The above solid was added to the deprotection solution (TFA:Tis:PhOH:3-Mpa:H2O=90:4:2.5:2.2:1) and reacted for 120 min. After the reaction was completed, the liquid was added to diethyl ether, mixed, filtered and washed, and dried to obtain impurity I.

[0046] The structure of the impurity of Formula I was confirmed by mass spectrometry (MS / MS) secondary fragment structure and proton NMR spectroscopy. The matching diagrams of the MS / MS secondary fragments of the synthesized impurity reference standard with the theoretical secondary fragments of the esterification impurity at position 12 are shown below. Figure 5 As shown in Table 3. The acquisition conditions for 1H NMR were as follows: the peptide was dissolved in 500 μL of D₂O, transferred to an NMR tube, and 1H NMR, 13C NMR, 1H-13C HMSQC, 1H-13C HMBC, noesy, and tocsy were acquired. The identification results of the NMR chemical shifts are as follows: Figure 6 As shown. Based on the above results, it can be concluded that an impurity with the structure of formula I was successfully prepared.

[0047] Example 3: Preparation of 8-position deamination and esterification impurities (Formula II impurities) by solid-phase synthesis method There is a deamination and esterification reaction between terlipressin and the non-reducing sugar mannitol, which occurs at the 8th position of asparagine.

[0048] The preparation of the mannitol esterified impurity involves first synthesizing the terlipressin peptide chain stepwise on a solid-phase support, and then reacting it with mannitol, including the following steps: (1) Weigh Fmoc amide AM resin and place it in a reaction tube, then add DCM to swell it. After swelling, add DBLK solution (20% pip / DMF solution) for deprotection reaction for 20 min. After the reaction, add DMF to wash. After washing, add Fmoc-Gly-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF to wash three times, then add DMF, acetic anhydride, and NMM (5 eq each) for acetylation reaction for 20 min. After the reaction, add DMF to wash.

[0049] (2) Add DBLK solution (20% pip / DMF solution) for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Lys(Boc)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and proceeded to the next condensation cycle.

[0050] (3) Add DBLK solution (20% pip / DMF solution) for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Pro-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and entered the next condensation cycle.

[0051] (4) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Cys(Trt)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. Take the resin and test it by Kaiser method. If the resin is colorless, proceed to the next condensation cycle.

[0052] (5) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Asp(OAll)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and entered the next condensation cycle.

[0053] (6) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Gln(Trt)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and entered the next condensation cycle.

[0054] (7) Add DBLK solution (20% pip / DMF solution) for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Phe-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin is tested by Kaiser method. If the resin is colorless, proceed to the next condensation cycle.

[0055] (8) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Tyr(tBu)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. The resin was tested by Kaiser method. The resin was colorless and entered the next condensation cycle.

[0056] (9) Add DBLK solution (20% pip / DMF solution) for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Cys(Trt)-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. Take the resin and test it by Kaiser method. If the resin is colorless, proceed to the next condensation cycle.

[0057] (10) Add DBLK solution for deprotection reaction for 20 min. After the reaction, add DMF for washing. After washing, add Fmoc-Gly-Gly-Gly-OH, HOBT, DIC (2 eq each) and DMF for condensation reaction for 90 min. After the reaction, add DMF for washing. Take the resin and test it by Kaiser method. If the resin is colorless, proceed to the next condensation cycle.

[0058] (11) Add tetraphenylphosphine palladium (2 eq) and DCM and react for 120 min. After the reaction is complete, add DMF to wash. Add mannitol, HOBT, DIC (2 eq each) and DMF and condense for 90-150 min. After the reaction is complete, dry the resin and add DMF to wash. Add DBLK solution and deprotect the resin for 20 min. After the reaction is complete, add DMF and diethyl ether to wash. After washing, dry the resin. (12) Add the peptide resin to the cutting solution (TFA:Tis:PhOH:3-Mpa:H2O=90:4:2.5:2.2:1) and react for 120 min. After the reaction is completed, filter the liquid and add it to diethyl ether. Mix, filter and wash, wash and dry to obtain crude product, and obtain impurity II.

[0059] The structure of the impurity of Formula II was confirmed by mass spectrometry (MS / MS) secondary fragment structure and proton NMR spectroscopy. The matching diagrams of the MS / MS secondary fragments of the synthetic impurity reference standard with the theoretical secondary fragments of the esterification impurity at position 8 are shown below. Figure 7 As shown in Table 4. The acquisition conditions for 1H NMR were as follows: the peptide was dissolved in 500 μL of D₂O, transferred to an NMR tube, and 1H NMR, 13C NMR, 1H-13C HMSQC, 1H-13C HMBC, noesy, and tocsy were acquired. The NMR chemical shift identification results are as follows: Figure 8 As shown. Based on the above results, it can be concluded that an impurity with the structure of formula II was prepared.

[0060] Example 4: Impurity Detection of Terlipressin Injection Formulation (1) Solution preparation: ① Preparation of impurity reference standard stock solution (impurities of Formula I and Formula II): Weigh 5 mg of impurity reference standard, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the mark, and shake well.

[0061] ② Preparation of impurity reference solution (prepare fresh for use): Accurately measure 1 ml of impurity reference stock solution, place it in a 100 ml volumetric flask, dilute with water to the mark, shake well, then accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute with an aqueous solution containing 100 nM TCEP and 200 nM MIAM to the mark, shake well, and let stand in the dark for 2 minutes.

[0062] ③ Preparation of test solution (prepare fresh before use): Take one vial of test sample (0.86 mg terlipressin acetate, 10 mg mannitol, appropriate amount of hydrochloric acid to adjust pH value, add water for injection to 1 ml, freeze dry), dissolve in 1 ml of water, shake well, then accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with an aqueous solution containing 100 nM TCEP (tris(2-carboxyethyl)phosphine) and 200 nM IAM (iodoacetamide), shake well, and place in the dark for 2 minutes.

[0063] (2) Chromatographic detection The H-class ultra-high performance liquid chromatograph was used. Mobile phase A was acetonitrile-water (90:10) (containing 0.1% anhydrous formic acid), and mobile phase B was acetonitrile-water (60:40) (containing 0.1% anhydrous formic acid). Gradient elution was performed according to the program in Table 5. The chromatographic column was ACQUITY UPLC® Glycan BEH Amide (130 Å, 1.7 μm, 2.1 × 100 mm), the flow rate was 0.25 ml / min, the detection wavelength was 210 nm, and the injection volume was 1 μl.

[0064] (3) Test results Figure 9 The image shows the high-performance liquid chromatogram of the impurities represented by Formula I. Figure 10 The image shows the high-performance liquid chromatogram of the impurities represented by Formula II. Figure 11 This is the high-performance liquid chromatogram of the test sample. As can be seen from the figure, Figure 9 and Figure 10 Because the two impurities have similar structures and their high-performance liquid chromatograms are essentially identical, Formula I, Formula II, or a mixture of both can be used as impurity reference standards when detecting the total amount of terlipressin mannitol ester impurities. Furthermore, based on the above results, it can be reasonably inferred that the total amount of terlipressin mannitol ester impurities may also include deamination and esterification impurities of amino acids at other positions, such as position 7.

[0065] Figure 11 A peak identical to the impurity reference standard appeared at the retention time of 9.5 min, indicating that the formulation contains terlipressin mannitol ester impurity, with a content of 0.91%.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. A quality control and testing method for injectable terlipressin preparations, characterized in that, The content of the impurities was detected by ultra-high performance liquid chromatography, using impurities of formula I and / or formula II as impurity reference standards. The injectable terlipressin preparation contains terlipressin and mannitol. ， 。 2. The quality control and testing method as described in claim 1, characterized in that, The ultra-high performance liquid chromatography method includes the following steps: (1) Prepare impurity reference solution and test solution, wherein the test solution is terlipressin for injection; (2) In an ultra-high performance liquid chromatograph, a C18 column is used, and mobile phase A and mobile phase B are used to elute the impurity reference standard and the test sample at a flow rate of 0.1~0.5 ml / min. The detection wavelength is 200~400 nm to obtain a chromatogram and determine the impurity content.

3. The quality control and testing method as described in claim 1 or 2, characterized in that, The impurity content refers to the total content of esterified impurities of terlipressin and mannitol.

4. The quality control and testing method as described in claim 2, characterized in that, The mobile phase A is acetonitrile:water = 90:10 and contains 0.1% anhydrous formic acid, and the mobile phase B is acetonitrile:water = 60:40 and contains 0.1% anhydrous formic acid.

5. The quality control and testing method as described in claim 2, characterized in that, The gradient elution procedure is as follows: in the mobile phase, the initial gradient mobile phase B ratio is 0%, the mobile phase B ratio increases from 0% to 100% from 0 to 10 min, the mobile phase B ratio is 100% from 10 to 15 min, the mobile phase B ratio decreases from 100% to 0% from 15 to 16 min, and the mobile phase B ratio is 0% from 16 to 25 min.

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