CN104101678A - Method for determining content of five calcium salts in compound alpha-ketoacid tablet - Google Patents

Method for determining content of five calcium salts in compound alpha-ketoacid tablet Download PDF

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Publication number
CN104101678A
CN104101678A CN201310115260.9A CN201310115260A CN104101678A CN 104101678 A CN104101678 A CN 104101678A CN 201310115260 A CN201310115260 A CN 201310115260A CN 104101678 A CN104101678 A CN 104101678A
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mobile phase
calcium
phase
ketone
kinds
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郭志强
王宏玲
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Tianjin Jinyao Group Co Ltd
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Tianjin Jinyao Group Co Ltd
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Abstract

The invention provides a method for determining content of five calcium salts in a compound alpha-ketoacid tablet. According to the method, a reversed phase chromatographic column taking octadecylsilyl bonded silica as a filler is employed, the detection wavelength is 205-220 nm, a mobile phase is composed of an A phase and a B phase, the A mobile phase is an aqueous solution with pH of 2.0-4.0, the B mobile phase is acetonitrile, the volume ratio of the A mobile phase to the B mobile phase is 85:15-99:1, and pH of the A phase is adjusted by an acid or a buffer salt.

Description

The content assaying method of five kinds of calcium salts in a kind of α keto acid compound
technical field:
The present invention relates to the assay method of α-one valine calcium in α keto acid compound, α-one isoleucine calcium, alpha-keto-leucine-calcium, α-hydroxyl Methionine calcium salt and α-Ketophenylalanine Calcium content.
background technology:
α keto acid compound is to be gone on the market in Germany first than company by German Fresenius card for 1996, and clinically for the nutrition of Chronic Renal Failure Patients, prevention and treatment cause the protein metabolism infringement causing of lacking of proper care because of chronic renal insufficiency.The compound preparation of this product for containing five kinds of calcium salts (α-one valine calcium, α-one isoleucine calcium, alpha-keto-leucine-calcium, α-hydroxyl Methionine calcium salt and α-Ketophenylalanine Calcium) and 5 kinds of essential amino acids (1B acetate, L-threonine, L-Trp, L-Histidine, TYR).
Bibliographical information about the content assaying method of five kinds of calcium salts in α keto acid compound is few.Chen Ju is at " Chinese pharmacists ", 2010, the 13 volumes, the 10th phase reported and adopts Cation ion-exchange column to carry out chromatographic resolution to 5 kinds of calcium salts in α keto acid compound, and then carried out assay, based on chromatographic resolution principle be ion exclusion.But the drawback of this method is, requirement to chromatographic column is harsher, different Cation ion-exchange columns is larger on separating effect impact, the durability that is method is poor, in this section of document, compared Féraud door Rezex ROA-organicAcid (H+8%) (300mm*7.8mm) and Varian METACARB 87H (300mm*7.8mm) Hydrogen cation-exchange chromatography post to ketone in 2-ketoacid sheet for amino acid calcium and hydroxyl the separating effect for amino acid calcium.Result shows, ketone phenylamino acid calcium Rezex ROA-organicAcid (H+8%) (300mm*7.8mm) in post chromatographic peak seriously trail, affect quantitative accuracy, and in Varian METACARB 87H (300mm*7.8mm) post good separating effect, quantitatively accurate.In addition, Yang Dan etc. are at " Shenyang Pharmaceutical University's journal ", 2011, the 28th volume, the 4th phase was reported and adopts reverse-phase paired ion chromatography method to measure content and the related substance of keto-leucine calcium, but this document is also pointed out ion pair chromatogram and is existed chromatogram system to be not easy balance, the inherent defect of chromatogram poor reproducibility.
summary of the invention:
For overcoming above-mentioned defect of the prior art, it is separation chromatography post that this patent application is used the most general anti-phase C18 post at present, adopts the mobile phase system of reverse-phase chromatography, realize the assay of five seed amino acid calcium salts in α keto acid compound, method is easy, accurately, reproducible.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: adopt the reverse-phase chromatographic column that octadecylsilane chemically bonded silica is filling agent, detection wavelength is 205～220nm, mobile phase is by A phase and B phase composition, mobile phase A is the aqueous solution of pH2.0 ~ 4.0, Mobile phase B is acetonitrile, and mobile phase A/Mobile phase B (volume ratio) is 85:15 ~ 99:1, and the pH of A phase regulates by acid or buffer salt.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: preferred gradient wash-out, adopts gradient elution program as follows:
Time/minute Mobile phase A/Mobile phase B (volume ratio)
0 a
T1 a
T2 b
T3 b
T4 a
T5 a
a=89:11~94:6;?b=94:6~98:2,?b>a,?T1=3~6,?T2=?T1+0.5~2,?T3>35,T4>T3，T5>T4。
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: adopt gradient elution program as follows:
Time/minute Mobile phase A/Mobile phase B (volume ratio)
0 a
T1 a
T2 b
T3 b
T4 a
T5 a
a=90:10~93:7;?b=95:5~97:3,b>a,?T1=3~5,?T2=?T1+0.5~2,?T3≥35，T4>T3+2，T5>T4+8。
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: mobile phase A is the phosphoric acid buffer system of pH2.0 ~ 4.0, acetate buffer system or citric acid buffer system.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: mobile phase A is the phosphoric acid buffer system of pH2.2 ~ 3.0, acetate buffer system or citric acid buffer system.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: mobile phase A is the phosphoric acid buffer system of pH2.2 ~ 3.0.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: mobile phase A is the KH of pH2.5 2pO 4-H 3pO 4buffer solution.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: phosphoric acid buffer system, acetate buffer system or the citric acid buffer system of the preferred 0.05mol/L of mobile phase A pH2.0 ~ 4.0.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: the preferred 210nm of detection wavelength used.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: detected temperatures used is 25 DEG C ~ 40 DEG C.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: chromatographic condition is as follows: detection wavelength is 210nm; Detected temperatures is 30 DEG C; Adopt gradient elution program as follows: A phase: the KH of pH2.5 ± 0.05 2pO 4-H 3pO 4buffer solution B phase: acetonitrile
Time/minute Mobile phase A/Mobile phase B (volume ratio)
0 93:7
5 93:7
5.5 96:4
45 96:4
50 93:7
70 93:7
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: detection wavelength is 210nm; Detected temperatures is 30 DEG C; Mobile phase A is the H of pH 2.0 ± 0.05 3pO 4aqueous solution, Mobile phase B is acetonitrile, mobile phase A/Mobile phase B (volume ratio) is 98:2.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: detection wavelength is 210nm; Detected temperatures is 30 DEG C; Mobile phase A is pH 2.5 ± 0.05 KH 2pO 4-H 3pO 4aqueous solution, Mobile phase B is acetonitrile, mobile phase A/Mobile phase B (volume ratio) is 94:6.
The invention provides in a kind of α keto acid compound the assay method of four kinds of ketone for amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: chromatographic condition is as follows: detection wavelength is 210nm; Detected temperatures is 30 DEG C; Adopt gradient elution program as follows: A phase: the KH of pH2.5 ± 0.05 2pO 4-H 3pO 4buffer solution B phase: acetonitrile; Gradient condition:
Time (min) Mobile phase A/Mobile phase B (volume ratio)
0 90:10
3.5 90:10
4 95:5
40 95:5
60 90:10
70 90:10
The present invention is by the content of five kinds of calcium salts in external standard method calculation sample.
The pH that the present invention sets, uses through pH4.00 and 6.86 two and carries out corrected pH meter mensuration, may be subject to the various factors such as instrument, so the pH value of above-mentioned mobile phase A can be in the interior variation in existing value ± 0.05.
It is that the refining content obtaining of corresponding test sample is more than 99.5% standard items or work reference substance that the present invention mentions the reference substance of testing in analytical approach.
The analytical approach that the present invention mentions, its flow rate of mobile phase is set as the known general knowledge of those skilled in the art.Common scope is generally between 0.5ml/min to 2.0ml/min.
Find that through test temperature is little on the impact of this analytical approach, just can shorten the retention time at all peaks with the rising of temperature, we have selected 25 DEG C ~ 40 DEG C of common temperature ranges.
The concentration of finding buffer solution through test is little on the impact of this analytical approach, but the concentration of buffer solution is higher, can affect the life-span of chromatographic column, so the phosphoric acid buffer system of we preferred 0.05mol/L, acetate buffer system or citric acid buffer system.
" chemicals Quality Control Analysis method validation technological guidance principle " ([H] GPH5-1) that promulgate according to State Food and Drug Administration carries out methodology checking to above-mentioned analysis condition, proves that the method can meet the mensuration of ketone acid sheet content.
The high efficiency liquid phase method of the content detection of five seed amino acid calcium salts in α keto acid compound provided by the invention, tool has the following advantages:
(1) use the chromatographic column that common octadecylsilane chemically bonded silica is filling agent.
(2) common being easy to get of two media that mobile phase is selected, feasibility is high.
(3) operating process is simple and convenient, accurately, reproducible.
(4) applicability is good, can detect the content of five kinds of calcium salts in α keto acid compound simultaneously.
brief description of the drawings:
Fig. 1: embodiment 1-1 gained liquid chromatogram
Fig. 2: embodiment 1-2 gained liquid chromatogram
Fig. 3: embodiment 2-1 gained liquid chromatogram
Fig. 4: embodiment 2-2 gained liquid chromatogram
Embodiment
Below will by embodiment, the invention will be further described, these descriptions are not that content of the present invention is further limited.Person skilled should be understood that the replacement that is equal to that technical characterictic of the present invention is done, or improves accordingly, within still belonging to protection scope of the present invention.
embodiment first-class degree elution method
embodiment 1-1
Chromatographic condition:
High performance liquid chromatograph: waters 2469
Detecting device: UV-detector detects wavelength: 210 nm
Sample size: 20.0 μ l column temperatures: 30 DEG C
Flow velocity: 1.0 ml/min chromatographic columns: Agilent C18,5 μ m, 4.6 × 250 mm
Analytical balance: 100,000/, Sartorius BS21S
Mobile phase: H 3pO 4aqueous solution (pH 2.0 ± 0.05): acetonitrile=98:2
Operation:
The preparation of reference substance solution:
Get the about 0.430g of α-one valine calcium, the about 0.335g of α-one isoleucine calcium, the about 0.505g of alpha-keto-leucine-calcium, the about 0.295g of α-hydroxyl Methionine calcium salt, α-Ketophenylalanine Calcium approximately 0.340 g, above reference substance all calculates by dry product, accurately weighed, be placed in 100ml measuring bottle, the ultrasonic 15min that adds water makes its dissolving, be settled to groove, shake up.Precision pipettes above-mentioned solution 5ml and is placed in 50ml measuring bottle, is diluted with water to scale, shakes up, and to obtain final product.
The preparation of need testing solution:
Get 20 of this product, accurately weighed, calculate average sheet weight.Be ground into powder, precision weighing fine powder appropriate (being approximately equivalent to α-one valine calcium 0.430g), is placed in 100ml measuring bottle, and the ultrasonic 15min that adds water dissolves main ingredient, is settled to groove, shakes up, and filters; Discard just filtrate, precision pipettes subsequent filtrate 5ml and is placed in 50ml measuring bottle, is diluted with water to scale, shakes up, and to obtain final product.
Assay method:
According to high performance liquid chromatography (2010 editions two annex of Chinese Pharmacopoeia d) measure.
Precision measures reference substance solution and the each 20 μ l injection liquid chromatographies of need testing solution, records chromatogram; With calculated by peak area, obtain content by external standard method.
Testing result: as shown in Figure 1, relevant data is listed in the table below.
Sequence number Title Retention time (minute) The content (%) calculating
1 Keto-valine calcium 9.888 99.4
2 Hydroxyl Methionine calcium salt 15.237 101.1
3 Ketone isoleucine calcium 24.893 98.9
4 Keto-leucine calcium 30.057 98.4
5 Ketone Phenylalanine calcium salt 39.851 99.9
The equal >1.5 of degree of separation between adjacent peak.
embodiment 1-2
Chromatographic condition:
High performance liquid chromatograph: waters 2469
Detecting device: UV-detector detects wavelength: 210 nm
Sample size: 20.0 μ l column temperatures: 30 DEG C
Flow velocity: 1.0 ml/min chromatographic columns: Agilent C18,5 μ m, 4.6 × 250 mm
Analytical balance: 100,000/, Sartorius BS21S
Mobile phase: 0.2mol/L KH 2pO 4-H 3pO 4aqueous solution (pH 2.5 ± 0.05): acetonitrile=94:6
Operation: the preparation of reference substance solution, need testing solution and assay method: reference example 1-1.
Testing result: as shown in Figure 2, relevant data is listed in the table below.
Sequence number Title Retention time (minute) The content (%) calculating
1 Keto-valine calcium 7.241 99.5
2 Hydroxyl Methionine calcium salt 14.459 99.4
3 Ketone isoleucine calcium 16.079 101.4
4 Keto-leucine calcium 19.910 99.1
5 Ketone Phenylalanine calcium salt 33.205 98.9
The equal >1.5 of degree of separation between adjacent peak.
embodiment 1-3
Chromatographic condition:
High performance liquid chromatograph: waters 2469
Detecting device: UV-detector detects wavelength: 205 nm
Sample size: 20.0 μ l column temperatures: 40 DEG C
Flow velocity: 1.0 ml/min chromatographic columns: Agilent C18,5 μ m, 4.6 × 250 mm
Analytical balance: 100,000/, Sartorius BS21S
Mobile phase: HCl aqueous solution (pH 4.0 ± 0.05): acetonitrile=85:15
Operation: the preparation of reference substance solution, need testing solution and assay method: reference example 1-1.
Testing result: relevant data is listed in the table below.
Sequence number Title Retention time (minute) The content (%) calculating
1 Keto-valine calcium 6.3 99.8
2 Hydroxyl Methionine calcium salt 11.555 99.7
3 Ketone isoleucine calcium 12.853 100.5
4 Keto-leucine calcium 15.875 99.3
5 Ketone Phenylalanine calcium salt 25.466 99.0
The equal >1.5 of degree of separation between adjacent peak.
embodiment 1-4
Chromatographic condition:
High performance liquid chromatograph: waters 2469
Detecting device: UV-detector detects wavelength: 220 nm
Sample size: 20.0 μ l column temperatures: 25 DEG C
Flow velocity: 1.0 ml/min chromatographic columns: Agilent C18,5 μ m, 4.6 × 250 mm
Analytical balance: 100,000/, Sartorius BS21S
Mobile phase: HCl aqueous solution (pH 3.0 ± 0.05): acetonitrile=99:1
Operation: the preparation of reference substance solution, need testing solution and assay method: reference example 1-1.
Testing result: relevant data is listed in the table below.
Sequence number Title Retention time (minute) The content (%) calculating
1 Keto-valine calcium 11.441 99.7
2 Hydroxyl Methionine calcium salt 17.237 99.6
3 Ketone isoleucine calcium 26.125 99.8
4 Keto-leucine calcium 34.214 99.4
5 Ketone Phenylalanine calcium salt 43.556 99.5
The equal >1.5 of degree of separation between adjacent peak.
embodiment bis-linear gradient elution method
embodiment 2-1
Chromatographic condition:
High performance liquid chromatograph: waters 2469
Detecting device: UV-detector detects wavelength: 210 nm
Sample size: 20.0 μ l column temperatures: 30 DEG C
Flow velocity: 1.0 ml/min chromatographic columns: Agilent C18,5 μ m, 4.6 × 250 mm
Analytical balance: 100,000/, Sartorius BS21S
Mobile phase: A phase: 0.05 mol/L KH 2pO 4-H 3pO 4aqueous solution (pH 2.5 ± 0.05)
B phase: acetonitrile
Gradient condition:
Time/minute Mobile phase A/Mobile phase B (volume ratio)
0 93:7
5 93:7
5.5 96:4
45 96:4
50 93:7
70 93:7
Operation:
The preparation of reference substance solution:
Get the about 0.430g of α-one valine calcium, the about 0.335g of α-one isoleucine calcium, the about 0.505g of alpha-keto-leucine-calcium, the about 0.295g of α-hydroxyl Methionine calcium salt, α-Ketophenylalanine Calcium approximately 0.340 g, above reference substance all calculates by dry product, accurately weighed, be placed in 100ml measuring bottle, the ultrasonic 15min that adds water makes its dissolving, be settled to groove, shake up.Precision pipettes above-mentioned solution 5ml and is placed in respectively 50ml measuring bottle, is diluted with water to scale, shakes up, and to obtain final product.
The preparation of need testing solution:
Get 20 of this product, accurately weighed, calculate average sheet weight.Be ground into powder, precision weighing fine powder appropriate (being approximately equivalent to α-one for valine calcium 0.430g), is placed in 100ml measuring bottle, and the ultrasonic 15min that adds water dissolves main ingredient, is settled to groove, shakes up, and filters; Discard just filtrate, precision pipettes subsequent filtrate 5ml and is placed in 50ml measuring bottle, is diluted with water to scale, shakes up, and to obtain final product.
Assay method:
According to high performance liquid chromatography (2010 editions two annex of Chinese Pharmacopoeia d) measure.
Precision measures reference substance solution and the each 20 μ l injection liquid chromatographies of need testing solution, records chromatogram; With calculated by peak area, obtain content by external standard method.
Testing result: as shown in Figure 3, relevant data is listed in the table below.
Sequence number Title Retention time (minute) The content (%) calculating
1 Keto-valine calcium 6.646 101.1
2 Hydroxyl Methionine calcium salt 14.503 99.3
3 Ketone isoleucine calcium 16.624 98.9
4 Keto-leucine calcium 21.927 98.5
5 Ketone Phenylalanine calcium salt 40.756 98.8
The equal >1.5 of degree of separation between adjacent peak.
embodiment 2-2
Chromatographic condition:
High performance liquid chromatograph: waters 2469
Detecting device: UV-detector detects wavelength: 210 nm
Sample size: 20.0 μ l column temperatures: 30 DEG C
Flow velocity: 1.0 ml/min chromatographic columns: Agilent C18,5 μ m, 4.6 × 250 mm
Analytical balance: 100,000/, Sartorius BS21S
Mobile phase: A phase: 0.05 mol/L KH 2pO4-H 3pO4 aqueous solution (pH 2.5 ± 0.05)
B phase: acetonitrile
Gradient condition:
Time (min) Mobile phase A/Mobile phase B (volume ratio)
0 90:10
3.5 90:10
4 95:5
40 95:5
60 90:10
70 90:10
Operation: the preparation of reference substance solution, need testing solution and assay method: reference example 2-1.
Testing result: as shown in Figure 4, relevant data is listed in the table below.
Sequence number Title Retention time (minute) The content (%) calculating
1 Keto-valine calcium 5.595 100.2
2 Hydroxyl Methionine calcium salt 10.493 99.8
3 Ketone isoleucine calcium 12.628 99.9
4 Keto-leucine calcium 17.467 99.5
5 Ketone Phenylalanine calcium salt 33.276 99.5
The equal >1.5 of degree of separation between adjacent peak.
embodiment 2-3
Chromatographic condition:
High performance liquid chromatograph: waters 2469
Detecting device: UV-detector detects wavelength: 220 nm
Sample size: 20.0 μ l column temperatures: 40 DEG C
Flow velocity: 1.0 ml/min chromatographic columns: Agilent C18,5 μ m, 4.6 × 250 mm
Analytical balance: 100,000/, Sartorius BS21S
Mobile phase: A phase: 0.05mol/L CH 3cOONa-CH 3cOOH (pH 3.0 ± 0.05)
B phase: acetonitrile
Gradient condition:
Time (min) Mobile phase A/Mobile phase B (volume ratio)
0 90:10
5 90:10
5.5 97:3
40 97:3
60 90:10
70 90:10
Operation: the preparation of reference substance solution, need testing solution and assay method: reference example 2-1.
Testing result: relevant data is listed in the table below.
Sequence number Title Retention time (minute) The content (%) calculating
1 Keto-valine calcium 5.574 99.6
2 Hydroxyl Methionine calcium salt 14.623 100.3
3 Ketone isoleucine calcium 16.750 98.8
4 Keto-leucine calcium 22.195 98.5
5 Ketone Phenylalanine calcium salt 41.524 99.4
The equal >1.5 of degree of separation between adjacent peak.
embodiment 2-4
Chromatographic condition:
High performance liquid chromatograph: waters 2469
Detecting device: UV-detector detects wavelength: 205 nm
Sample size: 20.0 μ l column temperatures: 30 DEG C
Flow velocity: 1.0 ml/min chromatographic columns: Agilent C18,5 μ m, 4.6 × 250 mm
Analytical balance: 100,000/, Sartorius BS21S
Mobile phase: A phase: phosphate aqueous solution (pH 2.0 ± 0.05)
B phase: acetonitrile
Measure the gradient condition of content:
Operation: the preparation of reference substance solution, need testing solution and assay method: reference example 2-1.
Testing result: relevant data is listed in the table below.
Sequence number Title Retention time (minute) The content (%) calculating
1 Keto-valine calcium 6.909 99.4
2 Hydroxyl Methionine calcium salt 16.821 98.8
3 Ketone isoleucine calcium 19.931 99.1
4 Keto-leucine calcium 25.205 99.0
5 Ketone Phenylalanine calcium salt 45.003 99.3
The equal >1.5 of degree of separation between adjacent peak.
embodiment 2-5
Chromatographic condition:
High performance liquid chromatograph: waters 2469
Detecting device: UV-detector detects wavelength: 205 nm
Sample size: 20.0 μ l column temperatures: 25 DEG C
Flow velocity: 1.0 ml/min chromatographic columns: Agilent C18,5 μ m, 4.6 × 250 mm
Analytical balance: 100,000/, Sartorius BS21S
Mobile phase: A phase: 0.1 mol/L citric acid-Na 2hPO4 aqueous solution (pH 4.0 ± 0.05)
B phase: acetonitrile
Measure the gradient condition of content:
Operation: the preparation of reference substance solution, need testing solution and assay method: reference example 2-1.
Testing result: relevant data is listed in the table below.
Sequence number Title Retention time (minute) The content (%) calculating
1 Keto-valine calcium 5.537 99.4
2 Hydroxyl Methionine calcium salt 10.291 98.8
3 Ketone isoleucine calcium 12.382 99.1
4 Keto-leucine calcium 17.153 99.0
5 Ketone Phenylalanine calcium salt 33.002 99.3
The equal >1.5 of degree of separation between adjacent peak.

Claims (10)

  1. In a α keto acid compound four kinds of ketone for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, it is characterized in that: adopt the reverse-phase chromatographic column that octadecylsilane chemically bonded silica is filling agent, detection wavelength is 205～220nm, mobile phase is by A phase and B phase composition, mobile phase A is the aqueous solution of pH2.0 ~ 4.0 mutually, Mobile phase B is acetonitrile mutually, and the volume ratio of mobile phase A and Mobile phase B is 85:15 ~ 99:1, and the pH of A phase regulates by acid or buffer salt.
  2. 2. in α keto acid compound as claimed in claim 1, four kinds of ketone, for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, is characterized in that: gradient elution, adopts gradient elution program as follows:
    Time/minute The volume of the volume/Mobile phase B of mobile phase A 0 a T1 a T2 b T3 b T4 a T5 a
    a=89:11~94:6;?b=94:6~98:2,?b>a,?T1=3~6,?T2=?T1+0.5~2,?T3≥35,T4>T3，T5>T4。
  3. 3. in α keto acid compound as claimed in claim 2, four kinds of ketone, for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, is characterized in that: adopt gradient elution program as follows:
    Time/minute The volume of the volume/Mobile phase B of mobile phase A 0 a T1 a T2 b T3 b T4 a T5 a
    a=90:10~93:7;?b=95:5~97:3,b>a,?T1=3~5,?T2=?T1+0.5~2,?T3>35，T4>T3+2，T5>T4+8。
  4. 4. in the α keto acid compound as described in claim 1 ~ 3, four kinds of ketone, for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, is characterized in that: mobile phase A is the phosphoric acid buffer system of pH2.0 ~ 4.0, acetate buffer system or citric acid buffer system mutually.
  5. 5. in α keto acid compound as claimed in claim 4, four kinds of ketone, for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, is characterized in that: detection wavelength used is 210nm.
  6. 6. in α keto acid compound as claimed in claim 4, four kinds of ketone, for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, is characterized in that: detected temperatures used is 25 DEG C ~ 40 DEG C.
  7. 7. in α keto acid compound as claimed in claim 3, four kinds of ketone, for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, is characterized in that: chromatographic condition is as follows:
    Detection wavelength is 210nm;
    Detected temperatures is 30 DEG C;
    Adopt gradient elution program as follows:
    A phase: the KH of pH2.5 ± 0.05 2pO 4-H 3pO 4buffer solution
    B phase: acetonitrile
    Time/minute The volume of the volume/Mobile phase B of mobile phase A 0 93:7 5 93:7 5.5 96:4 45 96:4 50 93:7 70 93:7
    。
  8. 8. in α keto acid compound as claimed in claim 1, four kinds of ketone, for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, is characterized in that:
    Detection wavelength is 210nm;
    Detected temperatures is 30 DEG C;
    Mobile phase A is the H of pH 2.0 ± 0.05 3pO 4aqueous solution, Mobile phase B is acetonitrile, the volume ratio of mobile phase A and Mobile phase B is 98:2.
  9. 9. in α keto acid compound as claimed in claim 1, four kinds of ketone, for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, is characterized in that:
    Detection wavelength is 210nm;
    Detected temperatures is 30 DEG C;
    Mobile phase A is pH 2.5 ± 0.05 KH 2pO 4-H 3pO 4aqueous solution, Mobile phase B is acetonitrile, the volume ratio of mobile phase A and Mobile phase B is 94:6.
  10. 10. in α keto acid compound as claimed in claim 3, four kinds of ketone, for the assay method of amino-acid calcium and hydroxyl Methionine calcium salt content, is characterized in that: chromatographic condition is as follows:
    Detection wavelength is 210nm;
    Detected temperatures is 30 DEG C;
    Adopt gradient elution program as follows:
    A phase: the KH of pH2.5 ± 0.05 2pO 4-H 3pO 4buffer solution
    B phase: acetonitrile
    Gradient condition:
    Time (min) The volume of the volume/Mobile phase B of mobile phase A 0 90:10 3.5 90:10 4 95:5 40 95:5 60 90:10 70 90:10
    。
CN201310115260.9A 2013-04-03 2013-04-03 Method for determining content of five calcium salts in compound alpha-ketoacid tablet Pending CN104101678A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104316641A (en) * 2014-10-20 2015-01-28 华东理工大学 Method for detecting impurity content in ketophenylalanine calcium
CN105699502A (en) * 2016-05-09 2016-06-22 上海景峰制药有限公司 Method for detecting related substances of amino acid calcium components in compound alpha-ketoacid tablets
CN109991321A (en) * 2017-12-31 2019-07-09 天津药业研究院有限公司 A kind of HPLC analytical method of the phenylalanine in relation to substance
CN112697934A (en) * 2020-12-10 2021-04-23 武汉久安药业有限公司 Method for detecting content of pyroglutamic acid in compound amino acid injection

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005093056A1 (en) * 2004-03-29 2005-10-06 Kyowa Hakko Kogyo Co., Ltd. Process for producing dipeptide
CN102961378A (en) * 2012-11-27 2013-03-13 贵州信邦制药股份有限公司 Compound alpha-keto acid tablets and preparation method and detection method thereof
CN102988357A (en) * 2011-09-14 2013-03-27 北大方正集团有限公司 Compound alpha keto acid medicine composition and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005093056A1 (en) * 2004-03-29 2005-10-06 Kyowa Hakko Kogyo Co., Ltd. Process for producing dipeptide
CN102988357A (en) * 2011-09-14 2013-03-27 北大方正集团有限公司 Compound alpha keto acid medicine composition and preparation method thereof
CN102961378A (en) * 2012-11-27 2013-03-13 贵州信邦制药股份有限公司 Compound alpha-keto acid tablets and preparation method and detection method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DI ZHANG等: "A novel ion-pair RP-HPLC method for determination of five components in compound α-ketoacid tablets", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 *
胡世林等: "HPLC法测定复方α-酮酸片中五种氨基酸的含量", 《安徽医药》 *
蒋燕等: "HPLC法测定复方α-酮酸片中四种氨基酸的含量", 《中国医药导报》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104316641A (en) * 2014-10-20 2015-01-28 华东理工大学 Method for detecting impurity content in ketophenylalanine calcium
CN104316641B (en) * 2014-10-20 2015-11-18 华东理工大学 The detection method of impurity content in tung-oil coated urea
CN105699502A (en) * 2016-05-09 2016-06-22 上海景峰制药有限公司 Method for detecting related substances of amino acid calcium components in compound alpha-ketoacid tablets
CN109991321A (en) * 2017-12-31 2019-07-09 天津药业研究院有限公司 A kind of HPLC analytical method of the phenylalanine in relation to substance
CN112697934A (en) * 2020-12-10 2021-04-23 武汉久安药业有限公司 Method for detecting content of pyroglutamic acid in compound amino acid injection

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