Method for detecting content of impurity D-sodium lactate in sodium lactate Ringer's injection

Abstract

The invention discloses a method for detecting the content of impurity D-sodium lactate in a sodium lactate Ringer's injection, which comprises the following steps: adding a 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution into the sodium lactate Ringer's injection, and shaking uniformly; then immediately adding an N-hydroxysuccinimide solution, and uniformly shaking; carrying out shaking reaction for 20-40 minutes in a water bath at 35-45 DEG C; then adding methanol and uniformly shaking; then adding an R-1-Cbz-3-aminopyrrolidine hydrochloride solution, and uniformly shaking; and carrying out shaking reaction in a water bath at 35-45 DEG C for 1.5-2.5 hours to obtain the amide derivative. Sapphire C18 (250 * 4.6 mm, 5 [mu] m) is used as a chromatographic column, 234 nm is used as a detection wavelength, a monopotassium phosphate aqueous solution with a pH value of 6.5 is used as a mobile phase A, and methanol is used as a mobile phase B to carry out gradient elution so as to detect the content of D-sodium lactate in the sodium lactate Ringer's injection. The detection method has the advantages of high sensitivity, low cost, strong universality, almost no detection interference, good accuracy, high sensitivity, good specificity, stable formed derivative, short analysis time, cheap derivatization reagent and chromatographic column and the like, and can effectively overcome the defects in the prior art. The impurity D-sodium lactate in the sodium lactate Ringer's injection can be effectively controlled, and the quality and safety of the sodium lactate Ringer's injection are ensured.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical analysis chemistry, and relates to a detection method for the content of impurity D-sodium lactate in sodium lactate Ringer's injection, in particular to a detection method for separating and detecting the content of impurity D-sodium lactate in sodium lactate Ringer's injection by using a derivative-HPLC method. BACKGROUND

[0002] The raw material of the sodium lactate Ringer's injection is a sodium lactate solution, which is obtained by the reaction of lactic acid and sodium hydroxide. Lactic acid contains two optical isomers, D-lactic acid and L-lactic acid. Research generally believes that only L-lactic acid dehydrogenase which can rapidly metabolize L-lactic acid exists in the human body, and D-lactic acid dehydrogenase does not exist, so that the increase of the concentration of D-lactic acid in the blood plasma will cause acidosis. The national standard sodium lactate solution (GB25537-2010) stipulates that the amount of L-sodium lactate should be greater than or equal to 95% of the total sodium lactate, that is, the amount of D-sodium lactate should be less than 5%. Therefore, in order to control the quality and safety of the product, it is necessary to study the control of impurity D-sodium lactate in sodium lactate Ringer's injection; when calculated as sodium lactate, the amount of D-sodium lactate therein should be less than 5%.

[0003] At present, there is no literature report on the detection method for the content of impurity D-sodium lactate in sodium lactate Ringer's injection.

[0004] The currently reported methods for separating and detecting L-lactic acid / L-lactate and D-lactic acid / D-lactate chiral enantiomers in various samples (such as baijiu, pickles, fruit juice, fermentation broth, etc.) are mainly HPLC methods combined with chiral chromatographic columns. However, such methods use expensive chiral chromatographic columns, have shorter service life, lower loading capacity, poorer durability of the chromatographic column, higher requirements for the compatibility of the mobile phase, lower column efficiency, and slower separation speed; and because of the specific mobile phase, 6-8 hours or even longer time is required for instrument maintenance and column balancing before and after use, which is time-consuming, occupies the resources of the high-performance liquid chromatograph, and wastes resources and costs.

[0005] Few literatures report that L-lactic acid / L-lactate and D-lactic acid / D-lactate in samples are reacted with a derivatizing reagent and then detected by high-performance liquid chromatography.

[0006] CN115656385A reports that chiral amine is used as a derivatizing reagent, and UHPLC-MS / MS method is used to detect L-lactic acid and D-lactic acid in baijiu. However, this patent does not clearly indicate what specific chiral amine is used, and the mass spectrometer in this method is relatively expensive, has high maintenance cost, and has high detection cost. Many production enterprises do not have a mass spectrometer, and the detection is limited. It cannot be used for the detection of the content of D-sodium lactate in sodium lactate Ringer's injection.

[0007] Hisanori Hasegawa et al. published a paper (Anal Bioanal Chem. (2003) 377: 886-891) and Takeshi Fukushima et al. published a paper (Biomed. Chromatogr. 15: 189-195 (2001)) both of which used 4-nitro-7-piperazinobenzoxadiazole fluorescent reagent as a derivatization reagent, in the presence of triphenylphosphine, 2,2'-dipyridyl disulfide reagent, to determine D-lactic acid in serum or plasma by HPLC method. 4-nitro-7-piperazinobenzoxadiazole fluorescent reagent is relatively expensive, about 1000 yuan / 100 mg, and about 200 mg is required for detecting one sample. When the number of samples is large, more 4-nitro-7-piperazinobenzoxadiazole fluorescent reagent is required, and the cost is too high for long-term detection. It cannot be used for the determination of the content of D-sodium lactate in sodium lactate Ringer's injection. SUMMARY

[0008] The purpose of the present application is to provide a method for determining the content of impurity D-sodium lactate in sodium lactate Ringer's injection, which uses derivatization-high performance liquid chromatography for detection. The detection method has the advantages of high sensitivity, low cost, strong universality, almost no detection interference, good accuracy, high sensitivity, good specificity, stable derivative, short analysis time, cheap derivatization reagent and chromatographic column, etc. It can effectively solve the above-mentioned problems existing in the prior art. It can effectively control the impurity D-sodium lactate in sodium lactate Ringer's injection, and ensure the quality and safety of sodium lactate Ringer's injection.

[0009] The present inventors have conducted a large number of experiments and found that the reagents of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and R-1-Cbz-3-aminopyrrolidine hydrochloride react with L-sodium lactate and D-sodium lactate in sodium lactate Ringer's injection, respectively, to form derivatives with good stability, effective baseline separation between L-sodium lactate derivative and D-sodium lactate derivative, no interference from the matrix in sodium lactate Ringer's injection, good accuracy, strong specificity, high sensitivity, etc. It effectively solves the above-mentioned problems existing in the prior art.

[0010] In an embodiment of the present application, the present application provides a method for detecting the content of impurity D-sodium lactate in sodium lactate Ringer's injection, which uses derivatization-HPLC method for detection.

[0011] In an embodiment of the present application, the detection method of the present application, wherein the derivatization step is as follows:

[0012] Step 1: add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution into sodium lactate Ringer's injection, shake well; then immediately add N-hydroxysuccinimide solution, shake well; in 35-45℃ water bath, shake for 20-40 minutes, to obtain solution 1;

[0013] Step 2: add methanol into solution 1 obtained in step 1, shake well; then add R-1-Cbz-3-aminopyrrolidine hydrochloride solution, shake well; in 35-45℃ water bath, shake for 1.5-2.5 hours, to obtain amide derivative.

[0014] The diluent of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is pH 6.5 potassium dihydrogen phosphate aqueous solution, pH 6.5 sodium dihydrogen phosphate aqueous solution, preferably pH 6.5 potassium dihydrogen phosphate aqueous solution;

[0015] The diluent of the N-hydroxysuccinimide solution is pH 6.5 potassium dihydrogen phosphate aqueous solution, pH 6.5 sodium dihydrogen phosphate aqueous solution, preferably pH 6.5 potassium dihydrogen phosphate aqueous solution;

[0016] The diluent of the R-1-Cbz-3-aminopyrrolidine hydrochloride solution is pH 7.6 potassium dihydrogen phosphate aqueous solution, pH 7.6 sodium dihydrogen phosphate aqueous solution, preferably pH 7.6 potassium dihydrogen phosphate aqueous solution;

[0017] The 35-45℃ water bath is preferably 40℃ water bath;

[0018] The shaking reaction in step 1 for 20-40 minutes is preferably shaking reaction for 30 minutes;

[0019] The shaking reaction in step 2 for 1.5-2.5 hours is preferably shaking reaction for 2 hours.

[0020] In the embodiment of the present application, the detection method of the present application, wherein the HPLC method is a method using high performance liquid chromatograph, using ultraviolet-visible spectrophotometer (including diode array detector) as detector, using octadecylsilane bonded silica gel as filler, using pH 6.0-7.0 buffer salt aqueous solution as mobile phase A and methanol as mobile phase B and using the following gradient for separation and detection;

[0021] The gradient is Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 65-55 65-55 80 80 Mobile phase B (%) 20 20 35-45 35-45 20 20 ;

[0022] Preferably Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20 ;

[0023] The buffer saline solution with pH of 6.0-7.0 is a potassium dihydrogen phosphate aqueous solution with pH of 6.0-7.0, a dipotassium hydrogen phosphate aqueous solution with pH of 6.0-7.0, a sodium dihydrogen phosphate aqueous solution with pH of 6.0-7.0, a disodium hydrogen phosphate aqueous solution with pH of 6.0-7.0; preferably a potassium dihydrogen phosphate aqueous solution with pH of 6.0-7.0, and further preferably a potassium dihydrogen phosphate aqueous solution with pH of 6.5;

[0024] The chromatographic column filled with the octadecylsilane bonded silica gel is a Sapphire C18 (250*4.6mm.

[0025] In the embodiment of the present application, the detection method of the present application comprises the following steps:

[0026] (1) Solution preparation

[0027] Preparation of potassium dihydrogen phosphate aqueous solution (pH 6.5): 20 mmol / L potassium dihydrogen phosphate aqueous solution, and the pH is adjusted to 6.5 by using 0.1 mol / L sodium hydroxide solution;

[0028] Preparation of potassium dihydrogen phosphate aqueous solution (pH 7.6): 20 mmol / L potassium dihydrogen phosphate aqueous solution, and the pH is adjusted to 7.6 by using 0.1 mol / L sodium hydroxide solution;

[0029] Preparation of mobile phase A (potassium dihydrogen phosphate aqueous solution with pH of 6.5): 20 mmol / L potassium dihydrogen phosphate aqueous solution, and the pH is adjusted to 6.5 by using ammonia;

[0030] Preparation of mobile phase B: methanol;

[0031] Preparation of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is dissolved in potassium dihydrogen phosphate aqueous solution (pH 6.5), and diluted to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride 35 mg per 1 ml of solution;

[0032] Preparation of N-hydroxysuccinimide solution: N-hydroxysuccinimide is dissolved in potassium dihydrogen phosphate aqueous solution (pH 6.5), and diluted to N-hydroxysuccinimide 11.5 mg per 1 ml of solution;

[0033] Preparation of R-1-Cbz-3-aminopyrrolidine hydrochloride solution: R-1-Cbz-3-aminopyrrolidine hydrochloride is dissolved in potassium dihydrogen phosphate aqueous solution (pH 7.6), and diluted to R-1-Cbz-3-aminopyrrolidine hydrochloride 32 mg per 1 ml of solution;

[0034] Preparation of D-sodium lactate control solution: dissolve D-sodium lactate control solution in water and dilute to a solution containing 62 μg of D-sodium lactate control solution per 1 ml;

[0035] (2) Preparation of test solution

[0036] Preparation of solution 1: take 1 mole of sodium lactate Ringer injection solution, add 2.2 moles of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, shake well; immediately add 2.4 moles of N-hydroxysuccinimide solution, shake well; in a 40°C water bath, shake for 30 minutes to obtain solution 1;

[0037] Preparation of amide derivative solution: add methanol to solution 1 solution, shake well; add 1.5 moles of R-1-Cbz-3-aminopyrrolidine hydrochloride solution, shake well; add potassium dihydrogen phosphate aqueous solution (pH 7.6), shake well; in a 40°C water bath, shake for 2 hours to obtain amide derivative;

[0038] Preparation of test solution: dilute the amide derivative solution with potassium dihydrogen phosphate aqueous solution (pH 6.5) to a solution containing 0.00415 mmol / ml of sodium lactate amide derivative per 1 ml;

[0039] (3) Preparation of blank solution and control solution

[0040] Prepare the blank solution and D-sodium lactate control solution in the same way as the test solution;

[0041] Determination

[0042] Chromatographic column: Sapphire C18 (250*4.6mm, 5μm);

[0043] Detection wavelength: 234 nm;

[0044] Injection volume: 20 μl;

[0045] Column temperature: 35°C;

[0046] Flow rate: 1 ml / min;

[0047] Gradient elution program: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20 ;

[0048] Calculation method: peak area calculation by external standard method.

[0049] In an embodiment of the present application, the detailed steps of the detection method of the present application are as follows:

[0050] (1) Solution preparation

[0051] Preparation of potassium dihydrogen phosphate aqueous solution (pH 6.5): 20 mmol / L of potassium dihydrogen phosphate aqueous solution, pH is adjusted to 6.5 by 0.1 mol / L sodium hydroxide solution;

[0052] Preparation of potassium dihydrogen phosphate aqueous solution (pH 7.6): 20 mmol / L of potassium dihydrogen phosphate aqueous solution, pH is adjusted to 7.6 by 0.1 mol / L sodium hydroxide solution;

[0053] Preparation of mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5): 20 mmol / L of potassium dihydrogen phosphate aqueous solution, pH is adjusted to 6.5 by ammonia water;

[0054] Preparation of mobile phase B: take methanol;

[0055] Preparation of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution: dissolve 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in potassium dihydrogen phosphate aqueous solution (pH 6.5), and dilute to a solution containing 35 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride per 1 ml;

[0056] Preparation of N-hydroxysuccinimide solution: dissolve N-hydroxysuccinimide in potassium dihydrogen phosphate aqueous solution (pH 6.5), and dilute to a solution containing 11.5 mg of N-hydroxysuccinimide per 1 ml;

[0057] Preparation of R-1-Cbz-3-amino pyrrolidine hydrochloride solution: dissolve R-1-Cbz-3-amino pyrrolidine hydrochloride in potassium dihydrogen phosphate aqueous solution (pH 7.6), and dilute to a solution containing 32 mg of R-1-Cbz-3-amino pyrrolidine hydrochloride per 1 ml;

[0058] Preparation of D-sodium lactate control solution: dissolve D-sodium lactate control in water, and dilute to a solution containing 62 μg of D-sodium lactate control per 1 ml;

[0059] (2) Preparation of test solution

[0060] Take 3ml of sodium lactate Ringer's injection, placed in 20ml volumetric flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1ml, shake; immediately add N-hydroxysuccinimide solution 2ml, shake; in 40℃ water bath, shaking reaction 30min; add 4ml of methanol, shake; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1ml, shake; add potassium dihydrogen phosphate aqueous solution (pH7.6) 4ml, shake; in 40℃ water bath, shaking reaction 2h; finally with potassium dihydrogen phosphate aqueous solution (pH6.5) dilution to the mark, shake, get;

[0061] (3) Preparation of blank solution

[0062] Take 3ml of water, placed in 20ml volumetric flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1ml, shake; immediately add N-hydroxysuccinimide solution 2ml, shake; in 40℃ water bath, shaking reaction 30min; add 4ml of methanol, shake; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1ml, shake; add potassium dihydrogen phosphate aqueous solution (pH7.6) 4ml, shake; in 40℃ water bath, shaking reaction 2h; finally with potassium dihydrogen phosphate aqueous solution (pH6.5) dilution to the mark, shake, get;

[0063] (4) Preparation of reference solution

[0064] Take 3ml of D-sodium lactate reference solution, placed in 20ml volumetric flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1ml, shake; immediately add N-hydroxysuccinimide solution 2ml, shake; in 40℃ water bath, shaking reaction 30min; add 4ml of methanol, shake; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1ml, shake; add potassium dihydrogen phosphate aqueous solution (pH7.6) 4ml, shake; in 40℃ water bath, shaking reaction 2h; finally with potassium dihydrogen phosphate aqueous solution (pH6.5) dilution to the mark, shake, get;

[0065] (5) Measurement

[0066] Column: Sapphire C18(250*4.6mm,5μm);

[0067] Detection wavelength: 234nm;

[0068] Injection volume: 20μl;

[0069] Column temperature: 35℃;

[0070] Flow rate: 1ml / min;

[0071] Gradient elution procedure: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20

[0072] Calculation: Peak area by external standard. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 The figure represents the chromatogram of Blank Solution 3 under the item of "Specificity" in the method validation of Example 1.

[0074] Figure 2 The figure represents the chromatogram of Reference Solution under the item of "Specificity" in the method validation of Example 1.

[0075] Figure 3 The figure represents the chromatogram of 100% spiked sample solution under the item of "Specificity" in the method validation of Example 1.

[0076] Figure 4 The figure represents the chromatogram of sample solution under the item of "Specificity" in the method validation of Example 1.

[0077] Figure 5 The figure represents the chromatogram of Limit of Quantitation solution under the item of "Linearity and Range" in the method validation of Example 1. DETAILED DESCRIPTION

[0078] The present application is further illustrated by the following examples, which should not be construed as limiting the scope of the present application.

[0079] The reagents used in the following examples are commercially available.

[0080] Example One

[0081] 1.1 Detection method

[0082] 1.1.1 Solution preparation

[0083] Preparation of potassium dihydrogen phosphate aqueous solution (pH 6.5): 20 mmol / L potassium dihydrogen phosphate aqueous solution, adjust pH to 6.5 with 0.1 mol / L sodium hydroxide solution.

[0084] Preparation of potassium dihydrogen phosphate aqueous solution (pH 7.6): 20 mmol / L potassium dihydrogen phosphate aqueous solution, adjust pH to 7.6 with 0.1 mol / L sodium hydroxide solution.

[0085] Preparation of mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5): 20 mmol / L potassium dihydrogen phosphate aqueous solution, adjust pH to 6.5 with ammonia.

[0086] ​Preparation of mobile phase B: Take methanol.

[0087] 1. Preparation of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution: Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in aqueous potassium dihydrogen phosphate solution (pH 6.5) and dilute to a solution containing 35 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride per 1 ml.

[0088] 2. Preparation of N-hydroxysuccinimide solution: Dissolve N-hydroxysuccinimide in aqueous potassium dihydrogen phosphate solution (pH 6.5) and dilute to a solution containing 11.5 mg of N-hydroxysuccinimide per 1 ml.

[0089] 3. Preparation of R-1-Cbz-3-aminopyrrolidine hydrochloride solution: Dissolve R-1-Cbz-3-aminopyrrolidine hydrochloride in aqueous potassium dihydrogen phosphate solution (pH 7.6) and dilute to a solution containing 32 mg of R-1-Cbz-3-aminopyrrolidine hydrochloride per 1 ml.

[0090] 4. Preparation of D-sodium lactate control stock solution: Dissolve D-sodium lactate control in water and dilute to a solution containing 62 μg of D-sodium lactate control per 1 ml.

[0091] 5. Preparation of D-sodium lactate control stock solution: Dissolve D-sodium lactate control in water and dilute to a solution containing 186 μg of D-sodium lactate control per 1 ml.

[0092] 1.1.2 Preparation of test solution

[0093] Test solution: Take 3 ml of sodium lactate Ringer's injection, place in a 20 ml volumetric flask, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution 1 ml, shake well; immediately add N-hydroxysuccinimide solution 2 ml, shake well; shake in a 40°C water bath for 30 minutes; add 4 ml of methanol, shake well; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake well; add aqueous potassium dihydrogen phosphate solution (pH 7.6) 4 ml, shake well; shake in a 40°C water bath for 2 hours; finally dilute to the mark with aqueous potassium dihydrogen phosphate solution (pH 6.5), shake well, and obtain.

[0094] 1.1.3 Preparation of blank solution

[0095] Blank solution: take 3ml water, put in 20ml volumetric flask, add 1-ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride solution 1ml, shake well; then immediately add N-hydroxysuccinimide solution 2ml, shake well; in 40°C water bath, shake reaction for 30 minutes; then add 4ml methanol, shake well; then add R-1-Cbz-3- aminopyrrolidine hydrochloride solution 1ml, shake well; then add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4ml, shake well; in 40°C water bath, shake reaction for 2 hours; finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and it is obtained.

[0096] 1.1.4 Preparation of control solution

[0097] Control solution: take 3ml of D-sodium lactate control solution, put in 20ml volumetric flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1ml, shake well; then immediately add N-hydroxysuccinimide solution 2ml, shake well; in 40°C water bath, shake reaction for 30 minutes; then add 4ml methanol, shake well; then add R-1-Cbz-3- aminopyrrolidine hydrochloride solution 1ml, shake well; then add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4ml, shake well; in 40°C water bath, shake reaction for 2 hours; finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and it is obtained.

[0098] 1.1.5 Chromatographic conditions

[0099] Chromatographic column type: Sapphire C18 (250*4.6mm, 5μm); detection wavelength: 234nm; injection volume: 20μl; column temperature: 35°C; flow rate: 1ml / min;

[0100] Gradient elution program: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20 .

[0101] 1.1.6 Determination

[0102] Determination: take 20μl of the above blank solution, control solution and test solution (each 1 needle) into high performance liquid chromatograph, inject according to the above chromatographic conditions, and record chromatogram. Calculate by peak area according to external standard method. The content of D-lactic acid should not be more than 5%.

[0103] 1.2 Method validation

[0104] 1.2.1 Specificity

[0105] 1.2.1.1 Blank solution 1

[0106] Blank solution 1: take 8ml of mobile phase A and 2ml of mobile phase B, mix well, and it is obtained.

[0107] 1.2.1.2 Blank solution 2

[0108] Blank solution 2: Take 3 ml water in a 20 ml volumetric flask, add 3 ml of potassium dihydrogen phosphate aqueous solution (pH 6.5), add 4 ml of methanol, then add potassium dihydrogen phosphate aqueous solution (pH 7.6) 5 ml, and finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and it is ready.

[0109] 1.2.1.3 Blank solution 3

[0110] Blank solution 3: Prepare the blank solution under the item "1.1.3" in Example 1.

[0111] 1.2.1.4 Blank auxiliary solution

[0112] Blank auxiliary preparation: Take 3 g of sodium chloride, 0.15 g of potassium chloride, and 0.10 g of calcium chloride (as CaCl2·2H2O), accurately weigh, place in a 500 ml volumetric flask, dissolve with appropriate amount of water, and dilute to the mark with water, shake well, and it is ready.

[0113] Blank auxiliary solution: Take 3 ml of blank auxiliary, place in a 20 ml volumetric flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml, shake well; immediately add N-hydroxysuccinimide solution 2 ml, shake well; in a 40°C water bath, shake for 30 minutes; add 4 ml of methanol, shake well; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake well; add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake well; in a 40°C water bath, shake for 2 hours; finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and it is ready.

[0114] 1.2.1.5 Reference solution

[0115] Reference solution: Prepare the reference solution under the item "1.1.4" in Example 1.

[0116] 1.2.1.6 Test solution

[0117] Test solution: Prepare the test solution under the item "1.1.2" in Example 1.

[0118] 1.2.1.7 100% spiked test solution

[0119] 100% spiked sample solution: take 3 ml of sodium lactate Ringer's injection, put it in a 20 ml volumetric flask, then add 1 ml of D-sodium lactate control solution stock solution, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml, shake well; immediately add N-hydroxysuccinimide solution 2 ml, shake well; in a 40 °C water bath, shake for 30 minutes; then add 4 ml of methanol, shake well; then add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake well; then add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake well; in a 40 °C water bath, shake for 2 hours; finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and it is obtained.

[0120] 1.2.1.8 determination

[0121] Determination: take 20 μl of the above blank solution 1, blank solution 2, blank solution 3, blank excipient solution, control solution, sample solution, 100% spiked sample solution (1 needle each) into the high performance liquid chromatograph, sample according to the above chromatographic conditions, record the chromatogram. Among them, blank solution 1, blank solution 2, blank solution 3, blank excipient solution, sample solution should not interfere with the determination of D-sodium lactate derivative in the product.

[0122] 1.2.2 system suitability

[0123] 1.2.2.1 control solution

[0124] Control solution: prepare the control solution under the item "1.1.4" in Example One.

[0125] 1.2.2.2 100% spiked sample solution

[0126] 100% spiked sample solution: prepare the 100% spiked sample solution under the item "1.2.1.7" in Example One.

[0127] 1.2.2.3 determination

[0128] Determination: take 20 μl of the above control solution (5 needles), 100% spiked sample solution (1 needle) into the high performance liquid chromatograph, sample according to the above chromatographic conditions, record the chromatogram. Among them, the RSD of the peak area of D-sodium lactate derivative in the control solution continuously sampled for 5 needles should not be more than 2%, and the separation degree of D-sodium lactate derivative peak in 100% spiked sample solution and adjacent peak should be greater than 1.5.

[0129] 1.2.3 limit of quantification and limit of detection

[0130] 1.2.3.1 limit of quantification solution

[0131] Limit of quantitation solution: take appropriate amount of control solution, dilute with initial mobile phase to about 10 of signal-to-noise ratio of D-sodium lactate derivative peak.

[0132] 1.2.3.2 Detection limit solution

[0133] Detection limit solution: take appropriate amount of control solution, dilute with initial mobile phase to about 3 of signal-to-noise ratio of D-sodium lactate derivative peak.

[0134] 1.2.3.4 Determination

[0135] Determination: take 20 μl of the above limit of quantitation solution (6 needles) and detection limit solution (3 needles) into high performance liquid chromatograph, sample according to the above chromatographic conditions, and record chromatogram. Among them, the limit of quantitation solution is continuously sampled for 6 needles, the peak area RSD of D-sodium lactate derivative is not more than 20%, the signal-to-noise ratio is not less than 10, and the concentration of limit of quantitation solution should not be more than 10% of the limit concentration. The detection limit solution is continuously sampled for 3 needles, and the signal-to-noise ratio is not less than 3.

[0136] 1.2.4 Linearity and range

[0137] 1.2.4.1 Each linear solution

[0138] 30% linear solution: take 0.3 ml of D-sodium lactate control solution stock solution, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml into 20 ml volumetric flask, shake well; immediately add N-hydroxysuccinimide solution 2 ml, shake well; in 40°C water bath, shake and react for 30 minutes; then add 4 ml of methanol, shake well; then add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake well; then add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake well; in 40°C water bath, shake and react for 2 hours; finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and obtain.

[0139] 50% linear solution: take 0.5 ml of D-sodium lactate control solution stock solution, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml into 20 ml volumetric flask, shake well; immediately add N-hydroxysuccinimide solution 2 ml, shake well; in 40°C water bath, shake and react for 30 minutes; then add 4 ml of methanol, shake well; then add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake well; then add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake well; in 40°C water bath, shake and react for 2 hours; finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and obtain.

[0140] 100% linear solution: take 1 ml of D-lactic acid sodium control solution stock solution, placed in 20 ml flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml, shake; immediately add N-hydroxysuccinimide solution 2 ml, shake; in 40 °C water bath, shake reaction 30 min; add 4 ml of methanol, shake; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake; add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake; in 40 °C water bath, shake reaction 2 h; finally diluted to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake, get.

[0141] 150% linear solution: take 1.5 ml of D-lactic acid sodium control solution stock solution, placed in 20 ml flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml, shake; immediately add N-hydroxysuccinimide solution 2 ml, shake; in 40 °C water bath, shake reaction 30 min; add 4 ml of methanol, shake; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake; add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake; in 40 °C water bath, shake reaction 2 h; finally diluted to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake, get.

[0142] 200% linear solution: take 2 ml of D-lactic acid sodium control solution stock solution, placed in 20 ml flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml, shake; immediately add N-hydroxysuccinimide solution 2 ml, shake; in 40 °C water bath, shake reaction 30 min; add 4 ml of methanol, shake; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake; add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake; in 40 °C water bath, shake reaction 2 h; finally diluted to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake, get.

[0143] 1.2.4.2 determination

[0144] Determination: take each linear solution (1 needle) 20 μl injection into high performance liquid chromatograph, according to the above chromatographic conditions, record chromatogram. The linear range of 200% of the limit of quantification concentration ~ limit concentration, the regression linear correlation coefficient (r) should be greater than or equal to 0.999, the absolute value of the Y axis intercept and the response value of 100% limit concentration should be within 25%.

[0145] 1.2.5 accuracy

[0146] 1.2.5.1 control solution

[0147] Control solution: The control solution was prepared as described under "1.1.4" in Example 1.

[0148] 1.2.5.2 Blank solution

[0149] Blank solution: The blank solution was prepared as described under "1.1.2" in Example 1.

[0150] 1.2.5.3 Recovery solution

[0151] 50% spiked sample solution: Take 3 ml of sodium lactate Ringer's injection, put it in a 20 ml volumetric flask, then add 0.5 ml of D-sodium lactate control stock solution, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml, shake well; immediately add N-hydroxysuccinimide solution 2 ml, shake well; in a 40°C water bath, shake for 30 minutes; then add 4 ml of methanol, shake well; then add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake well; then add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake well; in a 40°C water bath, shake for 2 hours; finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and it is obtained. (Prepare 3 portions in parallel)

[0152] 100% spiked sample solution: The 100% spiked sample solution was prepared as described under "1.2.1.7" in Example 1. (Prepare 3 portions in parallel)

[0153] 150% spiked sample solution: Take 3 ml of sodium lactate Ringer's injection, put it in a 20 ml volumetric flask, then add 1.5 ml of D-sodium lactate control stock solution, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml, shake well; immediately add N-hydroxysuccinimide solution 2 ml, shake well; in a 40°C water bath, shake for 30 minutes; then add 4 ml of methanol, shake well; then add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake well; then add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake well; in a 40°C water bath, shake for 2 hours; finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and it is obtained. (Prepare 3 portions in parallel)

[0154] 1.2.5.4 Determination

[0155] Determination: Take 20 μl of the above control solution, blank solution, and each spiked sample solution (recovery solution) (each 1 needle) and inject them into the high performance liquid chromatograph, inject according to the above chromatographic conditions, and record the chromatogram. The recovery rates of the 150%, 100%, and 50% recovery solutions of the limit concentration are all between 80% and 120%, and the RSD of the recovery of the 9 recovery solutions is not more than 20%.

[0156] 1.2.6 Precision - Repeatability (Operator A)

[0157] 1.2.6.1 Blank Solution

[0158] Blank Solution: The blank solution was prepared as described under "1.1.3" in Example One.

[0159] 1.2.6.2 Reference Solution

[0160] Reference Solution: The reference solution was prepared as described under "1.1.4" in Example One.

[0161] 1.2.6.3 100% Spiked Test Solution

[0162] 100% Spiked Test Solution: The 100% spiked test solution was prepared as described under "1.2.1.7" in Example One. (6 replicates were prepared)

[0163] 1.2.6.4 Assay

[0164] Assay: The blank solution, the reference solution, and each of the 100% spiked test solutions (1 needle each) were each injected into the HPLC in 20 μl, and the chromatogram was recorded according to the above chromatographic conditions. The RSD of the content of D-sodium lactate in the 6 replicates of the 100% spiked test solution prepared by Operator A was not more than 20%.

[0165] 1.2.7 Precision - Intermediate Precision (Operator B)

[0166] 1.2.7.1 Blank Solution

[0167] Blank Solution: The blank solution was prepared as described under "1.1.3" in Example One.

[0168] 1.2.7.2 Reference Solution

[0169] Reference Solution: The reference solution was prepared as described under "1.1.4" in Example One.

[0170] 1.2.7.3 100% Spiked Test Solution

[0171] 100% Spiked Test Solution: The 100% spiked test solution was prepared as described under "1.2.1.7" in Example One. (6 replicates were prepared)

[0172] 1.2.7.4 Assay

[0173] Determination: 20 μl of the above-mentioned blank solution, control solution, each 100% spiked sample solution (each 1 needle) was injected into the high performance liquid chromatograph, sample was injected according to the above-mentioned chromatographic conditions, and chromatogram was recorded. Among them, the RSD of the content of D-sodium lactate in 6 parallel prepared 100% spiked sample solutions prepared by the operator B was not more than 20%; the RSD of the content of D-sodium lactate in 12 solutions was not more than 20%.

[0174] 1.2.8 Solution stability

[0175] 1.2.8.1 Control solution

[0176] Control solution: the control solution under the item "1.1.4" in Example One was prepared.

[0177] 1.2.8.2 100% spiked sample solution

[0178] 100% spiked sample solution: the 100% spiked sample solution under the item "1.2.1.7" in Example One was prepared.

[0179] 1.2.8.3 Determination

[0180] Determination: at room temperature, 20 μl of the control solution, each 100% spiked sample solution (each 1 needle) was taken at about 0 h, 2 h, 4 h, 8 h, 16 h, 24 h time points, injected into the high performance liquid chromatograph, sample was injected according to the above-mentioned chromatographic conditions, and chromatogram was recorded. Among them, the recovery rate of the control solution measured at each observation time point was in the range of 90-110%; the relative deviation of the content of D-sodium lactate in the 100% spiked sample solution measured at each observation time point and 0 h should not be more than 20%.

[0181] 1.2.9 Durability

[0182] 1.2.9.1 Blank solution

[0183] Blank solution: the blank solution under the item "1.1.3" in Example One was prepared.

[0184] 1.2.9.2 Control solution

[0185] Control solution: the control solution under the item "1.1.4" in Example One was prepared.

[0186] 1.2.9.3 100% spiked sample solution

[0187] 100% spiked sample solution: the 100% spiked sample solution under the item "1.2.1.7" in Example One was prepared.

[0188] 1.2.9.4 Determination

[0189] Determination: Take the above-mentioned blank solution, control solution, each 100% spiked test sample solution (1 needle each) 20 μl into the high performance liquid chromatograph, according to the above-mentioned chromatographic conditions, record the chromatogram.

[0190] 1.3 Method validation results statistics

[0191] Example Two

[0192] Potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution preparation solution were prepared as in "Example One".

[0193] Preparation of test sample solution, blank solution, control solution, and 100% spiked test sample solution, except that the water bath temperature was 35°C, and the rest was the same as in "Example One".

[0194] Chromatographic conditions were the same as in "Example One".

[0195] Determination: Take the above-mentioned blank solution, control solution, test sample solution (1 needle each) 20 μl into the high performance liquid chromatograph, according to the above-mentioned chromatographic conditions, record the chromatogram.

[0196] Determination results: The recovery rate of this experiment was 89.3%.

[0197] Example Three

[0198] Potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution preparation solution were prepared as in "Example One".

[0199] Preparation of test solution, blank solution, control solution, 100% spiked test solution. Except that "the water bath temperature is 45°C", the rest is the same as "Example 1".

[0200] The chromatographic conditions are the same as "Example 1".

[0201] Determination: inject 20 μl of the above-mentioned blank solution, control solution, test solution (each 1 needle) into the high performance liquid chromatograph, sample according to the above-mentioned chromatographic conditions, and record the chromatogram.

[0202] Determination result: the recovery rate of this experiment is 90.4%.

[0203] Example Four

[0204] Preparation of potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution stock solution are the same as "Example 1".

[0205] Preparation of test solution, blank solution, control solution, 100% spiked test solution. Except that "add 2 ml of N-hydroxysuccinimide solution immediately, shake well; shake in a 40°C water bath for 20 minutes", the rest is the same as "Example 1".

[0206] The chromatographic conditions are the same as "Example 1".

[0207] Determination: inject 20 μl of the above-mentioned blank solution, control solution, test solution (each 1 needle) into the high performance liquid chromatograph, sample according to the above-mentioned chromatographic conditions, and record the chromatogram.

[0208] Determination result: the recovery rate of this experiment is 82.5%.

[0209] Example Five

[0210] Preparation of potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution stock solution are the same as "Example 1".

[0211] Preparation of test solution, blank solution, control solution and 100% spiked test solution. Except for "add 2 ml of N-hydroxysuccinimide solution, shake well; in 40°C water bath, shake for 40 minutes", the rest is the same as "Example 1".

[0212] Chromatographic conditions are the same as "Example 1".

[0213] Determination: inject 20 μl of the above-mentioned blank solution, control solution and test solution (each 1 needle) into high performance liquid chromatograph, sample according to the above-mentioned chromatographic conditions, and record chromatogram.

[0214] Determination result: the recovery rate of this experiment is 101.4%.

[0215] Example 6

[0216] Preparation of potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution and D-sodium lactate control solution reserve solution are the same as "Example 1".

[0217] Preparation of test solution, blank solution, control solution and 100% spiked test solution. Except for "add 1 ml of R-1-Cbz-3-aminopyrrolidine hydrochloride solution, shake well; add 4 ml of potassium dihydrogen phosphate aqueous solution (pH 7.6), shake well; in 40°C water bath, shake for 1.5 hours", the rest is the same as "Example 1".

[0218] Chromatographic conditions are the same as "Example 1".

[0219] Determination: inject 20 μl of the above-mentioned blank solution, control solution and test solution (each 1 needle) into high performance liquid chromatograph, sample according to the above-mentioned chromatographic conditions, and record chromatogram.

[0220] Determination result: the recovery rate of this experiment is 94.7%.

[0221] Example 7

[0222] Preparation of potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution and D-sodium lactate control solution reserve solution are the same as "Example 1".

[0223] Preparation of test solution, blank solution, control solution, 100% spiked test solution, except that "add 1 ml of R-1-Cbz-3-amino pyrrolidine hydrochloride solution, shake well; add 4 ml of potassium dihydrogen phosphate aqueous solution (pH 7.6), shake well; shake the reaction in a 40°C water bath for 2.5 hours" and the rest is the same as "Example 1".

[0224] Chromatographic conditions are the same as "Example 1".

[0225] Determination: inject 20 μl of the above-mentioned blank solution, control solution, test solution (each 1 needle) into the high performance liquid chromatograph, inject according to the above-mentioned chromatographic conditions, and record the chromatogram.

[0226] Determination result: the recovery rate of this experiment is 100.8%.

[0227] Example Eight

[0228] 8.1.1 Solution preparation

[0229] Preparation of sodium dihydrogen phosphate aqueous solution (pH 6.5): 20 mmol / L of sodium dihydrogen phosphate aqueous solution is adjusted to pH 6.5 with 0.1 mol / L sodium hydroxide solution.

[0230] Preparation of sodium dihydrogen phosphate aqueous solution (pH 7.6): 20 mmol / L of sodium dihydrogen phosphate aqueous solution is adjusted to pH 7.6 with 0.1 mol / L sodium hydroxide solution.

[0231] Preparation of mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5): 20 mmol / L of potassium dihydrogen phosphate aqueous solution is adjusted to pH 6.5 with ammonia.

[0232] Preparation of mobile phase B: take methanol.

[0233] Preparation of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution: dissolve 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in sodium dihydrogen phosphate aqueous solution (pH 6.5) and dilute to a solution containing 35 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride per 1 ml.

[0234] Preparation of N-hydroxysuccinimide solution: dissolve N-hydroxysuccinimide in sodium dihydrogen phosphate aqueous solution (pH 6.5) and dilute to a solution containing 11.5 mg of N-hydroxysuccinimide per 1 ml.

[0235] Preparation of R-1-Cbz-3-aminopyrrolidine hydrochloride solution: Dissolve R-1-Cbz-3-aminopyrrolidine hydrochloride in aqueous sodium dihydrogen phosphate solution (pH 7.6) and dilute to a solution containing 32 mg of R-1-Cbz-3-aminopyrrolidine hydrochloride per 1 ml.

[0236] Preparation of D-sodium lactate control stock solution: Dissolve D-sodium lactate control in water and dilute to a solution containing 62 μg of D-sodium lactate control per 1 ml.

[0237] Preparation of D-sodium lactate control stock solution: Dissolve D-sodium lactate control in water and dilute to a solution containing 62 μg of D-sodium lactate control per 1 ml.

[0238] 8.1.2 Preparation of test solution

[0239] Test solution: Take 3 ml of sodium lactate Ringer's injection, place in a 20 ml volumetric flask, add 1 ml of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, shake well; immediately add 2 ml of N-hydroxysuccinimide solution, shake well; shake in a 40°C water bath for 30 minutes; add 4 ml of methanol, shake well; add 1 ml of R-1-Cbz-3-aminopyrrolidine hydrochloride solution, shake well; add 4 ml of aqueous sodium dihydrogen phosphate solution (pH 7.6), shake well; shake in a 40°C water bath for 2 hours; finally dilute to the mark with aqueous sodium dihydrogen phosphate solution (pH 6.5), shake well, and obtain.

[0240] 8.1.3 Preparation of blank solution

[0241] Blank solution: Take 3 ml of water, place in a 20 ml volumetric flask, add 1 ml of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, shake well; immediately add 2 ml of N-hydroxysuccinimide solution, shake well; shake in a 40°C water bath for 30 minutes; add 4 ml of methanol, shake well; add 1 ml of R-1-Cbz-3-aminopyrrolidine hydrochloride solution, shake well; add 4 ml of aqueous sodium dihydrogen phosphate solution (pH 7.6), shake well; shake in a 40°C water bath for 2 hours; finally dilute to the mark with aqueous sodium dihydrogen phosphate solution (pH 6.5), shake well, and obtain.

[0242] 8.1.4 Preparation of control solution

[0243] Control solution: take 3 ml of D-sodium lactate control solution, put it in a 20 ml flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml, shake well; immediately add N-hydroxysuccinimide solution 2 ml, shake well; in 40°C water bath, shake reaction for 30 minutes; add 4 ml of methanol, shake well; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake well; add sodium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake well; in 40°C water bath, shake reaction for 2 hours; finally dilute to the mark with sodium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and it is obtained.

[0244] 8.1.5 100% spiked test solution

[0245] 100% spiked test solution: take 3 ml of sodium lactate Ringer's injection, put it in a 20 ml flask, then add 1 ml of D-sodium lactate control solution, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1 ml, shake well; immediately add N-hydroxysuccinimide solution 2 ml, shake well; in 40°C water bath, shake reaction for 30 minutes; add 4 ml of methanol, shake well; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1 ml, shake well; add sodium dihydrogen phosphate aqueous solution (pH 7.6) 4 ml, shake well; in 40°C water bath, shake reaction for 2 hours; finally dilute to the mark with sodium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and it is obtained.

[0246] 8.1.6 Chromatographic conditions

[0247] Chromatographic column type: Sapphire C18 (250*4.6mm, 5μm); detection wavelength: 234nm; injection volume: 20μl; column temperature: 35°C; flow rate: 1ml / min;

[0248] Gradient elution program: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20 .

[0249] 8.1.7 Determination

[0250] Determination: take 20μl of the above blank solution, control solution, test solution (each 1 needle) and inject into high performance liquid chromatograph, sample according to the above chromatographic conditions, record chromatogram.

[0251] 8.1.8 Determination results

[0252] The recovery rate of this experiment is 85.7%.

[0253] Example Nine

[0254] Potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution stock solution were prepared as described in "Example One".

[0255] Preparation of mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.0): 20 mmol / L potassium dihydrogen phosphate aqueous solution, pH adjusted to 6.0 with ammonia water.

[0256] Preparation of test sample solution, blank solution, control solution, and 100% spiked test sample solution was as described in "Example One".

[0257] Chromatographic conditions: column type: Sapphire C18 (250*4.6 mm, 5 μm); detection wavelength: 234 nm; injection volume: 20 μl; column temperature: 35 °C; flow rate: 1 ml / min;

[0258] Gradient elution program: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20 .

[0259] Determination: 20 μl of the above-mentioned blank solution, control solution, and test sample solution (each 1 needle) was injected into the high performance liquid chromatograph, and the chromatogram was recorded under the above-mentioned chromatographic conditions.

[0260] Determination result: the recovery rate of this experiment was 95.7%.

[0261] Example Ten

[0262] Potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution stock solution were prepared as described in "Example One".

[0263] Preparation of mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 7.0): 20 mmol / L potassium dihydrogen phosphate aqueous solution, pH adjusted to 7.0 with ammonia water.

[0264] Preparation of test sample solution, blank solution, control solution, and 100% spiked test sample solution was as described in "Example One".

[0265] Chromatographic conditions: Column type: Sapphire C18 (250*4.6 mm, 5 μm); detection wavelength: 234 nm; injection volume: 20 μl; column temperature: 35 °C; flow rate: 1 ml / min;

[0266] Gradient elution program: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20 .

[0267] Determination: Take the above-mentioned blank solution, control solution, test solution (each 1 needle) 20 μl into the high performance liquid chromatograph, sample according to the above chromatographic conditions, record the chromatogram.

[0268] Determination results: The recovery rate of this experiment is 96.2%.

[0269] Example Eleven

[0270] Potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution stock solution are prepared as in "Example One".

[0271] Preparation of mobile phase A (potassium hydrogen phosphate aqueous solution with pH 6.5): 20 mmol / L potassium hydrogen phosphate aqueous solution, adjust pH to 6.5 with ammonia water.

[0272] Preparation of test solution, blank solution, control solution, and 100% spiked test solution is the same as "Example One".

[0273] Chromatographic conditions: Column type: Sapphire C18 (250*4.6 mm, 5 μm); detection wavelength: 234 nm; injection volume: 20 μl; column temperature: 35 °C; flow rate: 1 ml / min;

[0274] Gradient elution program: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20 .

[0275] Determination: Take the above-mentioned blank solution, control solution, test solution (each 1 needle) 20 μl into the high performance liquid chromatograph, sample according to the above chromatographic conditions, record the chromatogram.

[0276] Determination results: The recovery rate of this experiment is 86.8%.

[0277] Example Twelve

[0278] Potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution stock solution were prepared as described in "Example One".

[0279] Preparation of mobile phase A (sodium dihydrogen phosphate aqueous solution with pH 6.5): 20 mmol / L sodium dihydrogen phosphate aqueous solution, pH adjusted to 6.5 with ammonia water.

[0280] Preparation of test sample solution, blank solution, control solution, and 100% spiked test sample solution as described in "Example One".

[0281] Chromatographic conditions: column type: Sapphire C18 (250*4.6 mm, 5 μm); detection wavelength: 234 nm; injection volume: 20 μl; column temperature: 35 °C; flow rate: 1 ml / min;

[0282] Gradient elution program: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20 .

[0283] Determination: 20 μl of the above-mentioned blank solution, control solution, and test sample solution (each 1 needle) was injected into the high performance liquid chromatograph, and the chromatogram was recorded under the above-mentioned chromatographic conditions.

[0284] Determination result: the recovery rate of this experiment was 93.8%.

[0285] Example Thirteen

[0286] Potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution stock solution were prepared as described in "Example One".

[0287] Preparation of mobile phase A (sodium dihydrogen phosphate aqueous solution with pH 6.5): 20 mmol / L sodium dihydrogen phosphate aqueous solution, pH adjusted to 6.5 with ammonia water.

[0288] Preparation of test sample solution, blank solution, control solution, and 100% spiked test sample solution as described in "Example One".

[0289] Chromatographic conditions: Column type: Sapphire C18 (250*4.6 mm, 5 μm); detection wavelength: 234 nm; injection volume: 20 μl; column temperature: 35 °C; flow rate: 1 ml / min;

[0290] Gradient elution program: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 60 60 80 80 Mobile phase B (%) 20 20 40 40 20 20 .

[0291] Determination: Take 20 μl of the above-mentioned blank solution, control solution and test solution (each 1 needle) into the high performance liquid chromatograph, inject according to the above-mentioned chromatographic conditions, and record the chromatogram.

[0292] Determination result: The recovery rate of this experiment is 95.3%.

[0293] Example Fourteen

[0294] Potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, D-sodium lactate control solution stock solution, and D-sodium lactate control solution stock solution are prepared as in "Example One".

[0295] The preparation of test solution, blank solution, control solution and 100% spiked test solution is the same as "Example One".

[0296] Chromatographic conditions: Column type: Sapphire C18 (250*4.6 mm, 5 μm); detection wavelength: 234 nm; injection volume: 20 μl; column temperature: 35 °C; flow rate: 1 ml / min;

[0297] Gradient elution program: Time (min) 0 10 25 30 32 40 Mobile phase A (%) 80 80 65 65 80 80 Mobile phase B (%) 20 20 35 35 20 20 .

[0298] Determination: Take 20 μl of the above-mentioned blank solution, control solution and test solution (each 1 needle) into the high performance liquid chromatograph, inject according to the above-mentioned chromatographic conditions, and record the chromatogram.

[0299] Determination result: The recovery rate of this experiment is 76.3%.

[0300] Example Fifteen

[0301] Potassium dihydrogen phosphate aqueous solution (pH 6.5), potassium dihydrogen phosphate aqueous solution (pH 7.6), mobile phase A (potassium dihydrogen phosphate aqueous solution with pH 6.5), mobile phase B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, R-1-Cbz-3-aminopyrrolidine hydrochloride solution, preparation of D-sodium lactate control solution stock solution was the same as "Example One".

[0302] Preparation of test solution, blank solution, control solution, 100% spiked test solution was the same as "Example One".

[0303] Chromatographic conditions: column type: Sapphire C18 (250*4.6mm, 5μm); detection wavelength: 234nm; injection volume: 20μl; column temperature: 35℃; flow rate: 1ml / min;

[0304] Gradient elution program: ​ 0 10 25 30 32 40 ​ 80 80 55 55 80 80 ​ 20 20 45 45 20 20 .

[0305] Determination: 20μl of the above blank solution, control solution, test solution (each 1 needle) was injected into the high performance liquid chromatograph, and the chromatogram was recorded according to the above chromatographic conditions.

[0306] Determination result: the recovery rate of this experiment was 78.3%.

Claims

1. A method for detecting the content of impurity D-sodium lactate in sodium lactate Ringer's injection, characterized by The detection method adopts a derivative-HPLC detection method; comprising the following steps: Step 1: Add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution to the sodium lactate Ringer's injection, shake well; then immediately add N-hydroxysuccinimide solution, shake well; in a 35-45°C water bath, shake for 20-40 minutes to obtain solution 1; Step 2: Add methanol to the solution 1 obtained in step 1, shake well; then add R-1-Cbz-3-aminopyrrolidine hydrochloride solution, shake well; in a 35-45°C water bath, shake for 1.5-2.5 hours to obtain the amide derivative; Step 3: Use a high performance liquid chromatograph, take an ultraviolet-visible spectrophotometric detector (including a diode array detector) as the detector, use an octadecylsilane bonded silica gel as the stationary phase of the chromatographic column, take a buffer saline solution with a pH of 6.0-7.0 as the mobile phase A, and methanol as the mobile phase B, to separate and detect the amide derivative of D-sodium lactate obtained in step 2; Among them, the buffer saline solution with a pH of 6.0-7.0 is a potassium dihydrogen phosphate aqueous solution with a pH of 6.0-7.0, a dipotassium hydrogen phosphate aqueous solution with a pH of 6.0-7.0, a sodium dihydrogen phosphate aqueous solution with a pH of 6.0-7.0, or a disodium hydrogen phosphate aqueous solution with a pH of 6.0-7.0; The volume ratio of the mobile phase A to the mobile phase B is as follows: 。 2. The detection method according to claim 1, wherein, The diluent of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution is a potassium dihydrogen phosphate aqueous solution with a pH of 6.5 or a sodium dihydrogen phosphate aqueous solution with a pH of 6.5, preferably a potassium dihydrogen phosphate aqueous solution with a pH of 6.5; The diluent of the N-hydroxysuccinimide solution is a potassium dihydrogen phosphate aqueous solution with a pH of 6.5 or a sodium dihydrogen phosphate aqueous solution with a pH of 6.5, preferably a potassium dihydrogen phosphate aqueous solution with a pH of 6.5; The diluent of the R-1-Cbz-3-aminopyrrolidine hydrochloride solution is a potassium dihydrogen phosphate aqueous solution with a pH of 7.6 or a sodium dihydrogen phosphate aqueous solution with a pH of 7.6, preferably a potassium dihydrogen phosphate aqueous solution with a pH of 7.

6.

3. The detection method according to claim 2, wherein, The buffer saline solution with a pH of 6.0-7.0 is preferably a potassium dihydrogen phosphate aqueous solution with a pH of 6.0-7.0, and more preferably a potassium dihydrogen phosphate aqueous solution with a pH of 6.

5.

4. The detection method according to claim 3, wherein, The volume ratio of the mobile phase A to the mobile phase B is preferably as follows: 。 5. The detection method according to claim 4, wherein, The chromatographic column with the octadecylsilane bonded silica gel as the filler is a Sapphire C18 (250*4.6mm, 5μm).

6. The detection method according to claim 5, wherein, The 35-45°C water bath is preferably a 40°C water bath; The shaking reaction in step 1 is preferably a shaking reaction for 30 minutes; The shaking reaction in step 2 is preferably a shaking reaction for 2 hours.

7. The detection method according to claim 6, comprising the following steps: (1) Solution preparation Preparation of potassium dihydrogen phosphate aqueous solution (pH 6.5): 20mmol / L potassium dihydrogen phosphate aqueous solution, adjust pH to 6.5 with 0.1mol / L sodium hydroxide solution; Preparation of aqueous potassium dihydrogen phosphate solution (pH 7.6): 20 mmol / L aqueous potassium dihydrogen phosphate solution, adjust pH to 7.6 with 0.1 mol / L sodium hydroxide solution; Preparation of mobile phase A (aqueous potassium dihydrogen phosphate solution with pH 6.5): 20 mmol / L aqueous potassium dihydrogen phosphate solution, adjust pH to 6.5; Preparation of mobile phase B: take methanol; Preparation of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution: dissolve 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in aqueous potassium dihydrogen phosphate solution (pH 6.5) and dilute to a solution containing 35 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride per 1 ml; Preparation of N-hydroxysuccinimide solution: dissolve N-hydroxysuccinimide in aqueous potassium dihydrogen phosphate solution (pH 6.5) and dilute to a solution containing 11.5 mg of N-hydroxysuccinimide per 1 ml; Preparation of R-1-Cbz-3-aminopyrrolidine hydrochloride solution: dissolve R-1-Cbz-3-aminopyrrolidine hydrochloride in aqueous potassium dihydrogen phosphate solution (pH 7.6) and dilute to a solution containing 32 mg of R-1-Cbz-3-aminopyrrolidine hydrochloride per 1 ml; Preparation of D-sodium lactate control solution: dissolve D-sodium lactate control in water and dilute to a solution containing 62 μg of D-sodium lactate control per 1 ml; (2) Preparation of test solution Preparation of solution 1: take 1 molar number of sodium lactate Ringer's injection, add 2.2 molar number of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, shake well; immediately add 2.4 molar number of N-hydroxysuccinimide solution, shake well; in a 40℃ water bath, shake for 30 minutes to obtain solution 1; Preparation of amide derivative solution: add methanol to solution 1 solution, shake well; add 1.5 molar number of R-1-Cbz-3-aminopyrrolidine hydrochloride solution, shake well; add aqueous potassium dihydrogen phosphate solution (pH 7.6), shake well; in a 40℃ water bath, shake for 2 hours to obtain amide derivative; Preparation of test solution: dilute the amide derivative solution with aqueous potassium dihydrogen phosphate solution (pH 6.5) to a solution containing 0.00415 mmol / ml of sodium lactate amide derivative per 1 ml; (3) Preparation of blank solution and control solution Prepare the blank solution and D-sodium lactate control solution in the same way as the preparation of the test solution; (4) Determination Chromatographic column: Sapphire C18 (250*4.6mm, 5μm); Detection wavelength: 234 nm; Injection volume: 20 μl; Column temperature: 35℃; Flow rate: 1 ml / min; Gradient elution program: ； Calculation method: peak area calculation by external standard method.

8. The detection method according to claim 7, comprising the following steps: (1) Solution preparation Preparation of aqueous potassium dihydrogen phosphate solution (pH 6.5): 20 mmol / L aqueous potassium dihydrogen phosphate solution, adjust pH to 6.5 with 0.1 mol / L sodium hydroxide solution; Preparation of aqueous potassium dihydrogen phosphate solution (pH 7.6): 20 mmol / L aqueous potassium dihydrogen phosphate solution, adjust pH to 7.6 with 0.1 mol / L sodium hydroxide solution; Preparation of mobile phase A (aqueous potassium dihydrogen phosphate solution with pH 6.5): 20 mmol / L aqueous potassium dihydrogen phosphate solution, adjust pH to 6.5 with ammonia water; Preparation of mobile phase B: take methanol; Preparation of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution: dissolve 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in aqueous potassium dihydrogen phosphate solution (pH 6.5), and dilute to a solution containing 35 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride per 1 ml; Preparation of N-hydroxysuccinimide solution: dissolve N-hydroxysuccinimide in aqueous potassium dihydrogen phosphate solution (pH 6.5), and dilute to a solution containing 11.5 mg of N-hydroxysuccinimide per 1 ml; Preparation of R-1-Cbz-3-aminopyrrolidine hydrochloride solution: dissolve R-1-Cbz-3-aminopyrrolidine hydrochloride in aqueous potassium dihydrogen phosphate solution (pH 7.6), and dilute to a solution containing 32 mg of R-1-Cbz-3-aminopyrrolidine hydrochloride per 1 ml; Preparation of D-sodium lactate control solution: dissolve D-sodium lactate control in water, and dilute to a solution containing 62 μg of D-sodium lactate control per 1 ml; (2) Preparation of test solution Take 3 ml of sodium lactate Ringer's injection and place it in a 20 ml volumetric flask. Add 1 ml of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and shake well. Immediately add 2 ml of N-hydroxysuccinimide solution and shake well. Shake in a 40°C water bath for 30 minutes. Then add 4 ml of methanol and shake well. Add 1 ml of R-1-Cbz-3-aminopyrrolidine hydrochloride solution and shake well. Add 4 ml of aqueous potassium dihydrogen phosphate solution (pH 7.6) and shake well. Shake in a 40°C water bath for 2 hours. Finally, dilute to the mark with aqueous potassium dihydrogen phosphate solution (pH 6.5) and shake well to obtain the test solution. (3) Preparation of blank solution Take 3 ml of water and place it in a 20 ml volumetric flask. Add 1 ml of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and shake well. Immediately add 2 ml of N-hydroxysuccinimide solution and shake well. Shake in a 40°C water bath for 30 minutes. Then add 4 ml of methanol and shake well. Add 1 ml of R-1-Cbz-3-aminopyrrolidine hydrochloride solution and shake well. Add 4 ml of aqueous potassium dihydrogen phosphate solution (pH 7.6) and shake well. Shake in a 40°C water bath for 2 hours. Finally, dilute to the mark with aqueous potassium dihydrogen phosphate solution (pH 6.5) and shake well to obtain the blank solution. (4) Preparation of control solution Take 3ml of D-sodium lactate control solution in a 20ml flask, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution 1ml, shake well; immediately add N-hydroxysuccinimide solution 2ml, shake well; in 40℃ water bath, shake reaction for 30 minutes; add 4ml of methanol, shake well; add R-1-Cbz-3-aminopyrrolidine hydrochloride solution 1ml, shake well; add potassium dihydrogen phosphate aqueous solution (pH 7.6) 4ml, shake well; in 40℃ water bath, shake reaction for 2 hours; finally dilute to the mark with potassium dihydrogen phosphate aqueous solution (pH 6.5), shake well, and get; (5) Determination Column: Sapphire C18 (250*4.6mm, 5μm); Detection wavelength: 234nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 1ml / min; Gradient elution program: ； Calculation method: peak area calculation by external standard method.

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