CN105606724A - Method for assaying related substances in lysine acetate by high-performance liquid chromatography - Google Patents

Abstract

The invention relates to a method for assaying related substances in lysine acetate by high-performance liquid chromatography. According to the method, by taking octadecylsilane bonded silica gel as a chromatographic column and taking a sodium dihydrogen phosphate buffer solution and methanol-water as mobile phases, the contents of the related substances in the lysine acetate can be assayed, so that the masses of the related substances in the lysine acetate can be effectively controlled.

Description

A kind of method with related substance in high effective liquid chromatography for measuring Lysine Acetate

Technical field

The present invention relates to the determination techniques field of related substance in Lysine Acetate, particularly one high-efficient liquid phase colorSpectrometry is measured the method for related substance in Lysine Acetate.

Background technology

Lysine Acetate is one of essential amino acid, has the nutritive validity of supplementary protein, pharmaceutically can useIn preparation amino acid injection and oral formulations, also for preparing amino acid injection and oral formulations; The adjuvant of diureticsThing, treatment reduces the lead poisoning phenomenon causing because of chloride in blood; Can generate salt with acidic drug (as salicylic acid etc.) subtractsLight bad reaction.

Lysine Acetate belongs to Branchamin, its Chinese phonetic alphabet: CusuanLaiansuan, English name: LysineAcetate, proterties: this product is white crystals or crystalline powder; Almost odorless. This product is easily molten in water, in ethanol almostInsoluble. Chemical composition: it is L-2,6-diaminocaproic acid acetate. Press dry product and calculate, containing C₆H₁₄N₂O₂·C₂H₄O₂Must not lackIn 98.5%.

Related substance refers to the magazine of medicine except main composition, may be in building-up process, bring into cause raw material, centreBody, side reaction product, reagent, analyte, condensate, isomers, may be also in preparation production process or storage, transport,In use procedure, say hydrolysis products etc.

Impurity in Lysine Acetate is mainly other amino acidses, thereby affects quality and the purity of Lysine Acetate,Content is revised and enlarged in 2010 editions Chinese pharmacopoeia revisions, other amino acid whose detection method thin-layered chromatography, and with ninhydrin colour developing, be somebody's turn to doThe shortcoming of method can not be to the quantitative analysis of carrying out of all related impuritieses, and large molecule amino acid separating effect is undesirable, Bu NengjinThe comprehensive impurity analysis of row.

Due to amino acid self structure particularity, Lysine Acetate is without UV absorption and fluorescent emission feature. Therefore adoptGeneral analytical method is difficult to it to detect completely, need to derive it, generate have that UV, visible light absorbs orThe derivative of person's fluorescence response just can be analyzed mensuration.

The method that high performance liquid chromatography combines with various column front derivation technology has its distinctive superiority. O-phthalicAldehyde (OPA) is a kind of common derivative reagent, and under 3-mercaptopropionic acid exists, the amino acid reaction of alkali condition and one-level generates sulphurGeneration-2-alkyl iso-indoles, has stronger UV absorption, and the derivative time is short, method is easy, can complete online derivation operation. Therefore,For the analysis of related substance in Lysine Acetate, can adopt OPA column front derivation efficient liquid-phase chromatography method.

Summary of the invention

It is a kind of with efficient that technical problem to be solved by this invention is to provide for the existing deficiency of prior artThe method of related substance in liquid chromatography for measuring Lysine Acetate, Dichlorodiphenyl Acetate lysine production technology and quality control have fingerLead meaning.

Technical problem to be solved by this invention can be achieved through the following technical solutions:

By a method for related substance in high effective liquid chromatography for measuring Lysine Acetate, comprise the steps:

(1) chromatographic system establishment step

Set up chromatographic system according to following condition: chromatographic column: C18100*4.6mm, column temperature: 40 DEG C, detector: ultraviolet inspectionSurvey device, 336nm, flow velocity: 1.0ml/min, sampling volume: 10 μ l, derivatization reagent: OPA (OPA), mobile phase A:PH7.4～7.8 sodium dihydrogen phosphate buffer, Mobile phase B: methanol-water, wherein the volume ratio of methyl alcohol and water is 90:10;

(2) need testing solution preparation process

Precision takes in Lysine Acetate 50mg to 100ml measuring bottle, adds purified water appropriate ultrasonic quarter that makes to dissolve and be settled toDegree, filters, as need testing solution;

(3) test sample own control solution preparation process

The need testing solution of preparing from step (2), precision measures 1mL in 100mL volumetric flask, adds purified water and is diluted toScale, shakes up as test sample own control solution;

(4) detect analytical procedure:

Precision measures diluent, contrast solution and the need testing solution injection liquid chromatography respectively of 10 μ l, adopts before postDerivatization method, records chromatogram, in need testing solution chromatogram, as there is impurity chromatographic peak, records the peak area of each impurity, with mainComposition Self-control method is calculated impurity, and specific formula for calculation is as follows:

$\%impurityA=\frac{A_{spl}}{A_{std}}$

Note: A_splImpurity peak area in=sample solution; A_std=1.0% own control solution main peak peak area.

In a preferred embodiment of the invention, the preparation method of described pH7.4～7.8 sodium dihydrogen phosphate bufferBe: get sodium dihydrogen phosphate 7.15g and add water after 1000mL dissolving, with NaOH solution adjusting pH value to 7.4～7.8.

In a preferred embodiment of the invention, the gradient of described mobile phase A and Mobile phase B is as shown in table 1:

Table 1

Time	A％	B％
			0	90	10
16	80	20
			25	75	25
30	50	50
			33	20	80
35	0	100
			37	0	100
40	90	10
			45	90	10

。

Owing to having adopted technical scheme as above, relevant thing in high effective liquid chromatography for measuring Lysine Acetate for the present inventionThe advantages such as matter, can make Lysine Acetate and impurity obtain effective separation, and it is high to have precision, reproducible, workable,Dichlorodiphenyl Acetate lysine production technology and quality control have directive significance.

Detailed description of the invention

By a method for related substance in high effective liquid chromatography for measuring Lysine Acetate, comprise the steps:

(1) chromatographic system establishment step

Set up chromatographic system according to following condition: chromatographic column: C18100*4.6mm, column temperature: 40 DEG C, detector: ultraviolet inspectionSurvey device, 336nm, flow velocity: 1.0ml/min, sampling volume: 10 μ l, derivatization reagent: OPA (OPA), mobile phase A:PH7.4～7.8 sodium dihydrogen phosphate buffer, Mobile phase B: methanol-water, wherein the volume ratio of methyl alcohol and water is 90:10;

The preparation method of pH7.4～7.8 sodium dihydrogen phosphate buffer is: get the sodium dihydrogen phosphate 7.15g 1000mL that adds waterAfter dissolving, with NaOH solution adjusting pH value to 7.4～7.8.

The gradient of mobile phase A and Mobile phase B is as shown in table 1:

Table 1

Time	A％	B％
			0	90	10
16	80	20
			25	75	25
30	50	50
			33	20	80
35	0	100
			37	0	100
40	90	10
			45	90	10

。

(2) need testing solution preparation process

Precision takes in a certain batch of Lysine Acetate 50mg to 100ml measuring bottle, adds appropriate ultrasonic the making of purified water and dissolves alsoBe settled to scale, filter, as need testing solution;

(3) test sample own control solution preparation process

The need testing solution of preparing from step (2), precision measures 1mL in 100mL volumetric flask, adds purified water and is diluted toScale, shakes up as test sample own control solution;

(4) detect analytical procedure:

Precision measures diluent, contrast solution and the need testing solution injection liquid chromatography respectively of 10 μ l, adopts before postDerivatization method, records chromatogram, in need testing solution chromatogram, as there is impurity chromatographic peak, records the peak area of each impurity, with mainComposition Self-control method is calculated impurity, and specific formula for calculation is as follows:

$\%impurityA=\frac{A_{spl}}{A_{std}}$

Note: A_splImpurity peak area in=sample solution; A_std=1.0% own control solution main peak peak area.

In a certain batch of Lysine Acetate, impurity testing result is in table 2

Table 2: Lysine Acetate sample impurity testing result

From above detection Lysine Acetate, related substance testing result can be found out, according to method provided by the invention,Can make Lysine Acetate and impurity obtain effective separation, and it is high to have precision, reproducible, the advantage such as workable.

Claims (3)

1. by a method for related substance in high effective liquid chromatography for measuring Lysine Acetate, it is characterized in that, comprise as followsStep:

(1) chromatographic system establishment step

Set up chromatographic system according to following condition: chromatographic column: C18100*4.6mm, column temperature: 40 DEG C, detector: UV-detector,336nm, flow velocity: 1.0ml/min, sampling volume: 10 μ l, derivatization reagent: OPA (OPA), mobile phase A: pH7.4～7.8 sodium dihydrogen phosphate buffers, Mobile phase B: methanol-water, wherein the volume ratio of methyl alcohol and water is 90:10;

(2) need testing solution preparation process

Precision takes in Lysine Acetate 50mg to 100ml measuring bottle, adds the appropriate ultrasonic scale that makes to dissolve and be settled to of purified water, filterCross, as need testing solution;

(3) test sample own control solution preparation process

The need testing solution of preparing from step (2), precision measures 1mL in 100mL volumetric flask, and add purified water and be diluted to scale,Shake up as test sample own control solution;

(4) detect analytical procedure:

Precision measures diluent, contrast solution and the need testing solution injection liquid chromatography respectively of 10 μ l, adopts column front derivationMethod, records chromatogram, in need testing solution chromatogram, as there is impurity chromatographic peak, records the peak area of each impurity, uses principal componentSelf-control method is calculated impurity, and specific formula for calculation is as follows:

$\%impurityA=\frac{A_{spl}}{A_{std}}$

Note: A_splImpurity peak area in=sample solution; A_std=1.0% own control solution main peak peak area.

2. a kind of method with related substance in high effective liquid chromatography for measuring Lysine Acetate as claimed in claim 1, itsBe characterised in that, the preparation method of described pH7.4～7.8 sodium dihydrogen phosphate buffer is: get sodium dihydrogen phosphate 7.15g and add waterAfter 1000mL dissolves, with NaOH solution adjusting pH value to 7.4～7.8.

3. a kind of method with related substance in high effective liquid chromatography for measuring Lysine Acetate as claimed in claim 1, itsBe characterised in that, the gradient of described mobile phase A and Mobile phase B is as shown in table 1:

Table 1

Time A％ B％ 0 90 10 16 80 20 25 75 25 30 50 50 33 20 80 35 0 100 37 0 100 40 90 10 45 90 10

。