CN1749748A - Method for analysizing amino acid - Google Patents
Method for analysizing amino acid Download PDFInfo
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- CN1749748A CN1749748A CN 200510014478 CN200510014478A CN1749748A CN 1749748 A CN1749748 A CN 1749748A CN 200510014478 CN200510014478 CN 200510014478 CN 200510014478 A CN200510014478 A CN 200510014478A CN 1749748 A CN1749748 A CN 1749748A
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- 150000001413 amino acids Chemical class 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title abstract description 8
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 claims abstract description 35
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical class [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 12
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 103
- 239000000523 sample Substances 0.000 claims description 54
- 239000012086 standard solution Substances 0.000 claims description 28
- JRYTUFKIORWTNI-UHFFFAOYSA-N xylamidine Chemical group COC1=CC=CC(OC(C)CN=C(N)CC=2C=C(C)C=CC=2)=C1 JRYTUFKIORWTNI-UHFFFAOYSA-N 0.000 claims description 27
- 239000012488 sample solution Substances 0.000 claims description 25
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- 239000000243 solution Substances 0.000 claims description 18
- 238000013459 approach Methods 0.000 claims description 17
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 15
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- 238000010828 elution Methods 0.000 claims description 14
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 13
- 238000010829 isocratic elution Methods 0.000 claims description 11
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- 208000019423 liver disease Diseases 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 7
- 238000005191 phase separation Methods 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 239000003086 colorant Substances 0.000 claims description 6
- 238000009795 derivation Methods 0.000 claims description 6
- 238000010812 external standard method Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 6
- 238000012113 quantitative test Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims 2
- 239000012071 phase Substances 0.000 abstract description 29
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- 238000004458 analytical method Methods 0.000 abstract description 14
- 238000006243 chemical reaction Methods 0.000 abstract description 8
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- 238000011084 recovery Methods 0.000 description 52
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- 239000000126 substance Substances 0.000 description 16
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- -1 compound amino-acids Chemical class 0.000 description 12
- 238000012546 transfer Methods 0.000 description 11
- 238000005303 weighing Methods 0.000 description 9
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- 108010005233 alanylglutamic acid Proteins 0.000 description 6
- 108010044940 alanylglutamine Proteins 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000001816 cooling Methods 0.000 description 6
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- 239000004220 glutamic acid Substances 0.000 description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 6
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- 238000005259 measurement Methods 0.000 description 5
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- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
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Abstract
The amino acid analyzing method includes high efficiency liquid phase chromatographic separation of the 2, 4-dinitro fluoro benzene derivative of amino acid in C18 column with pH 2-3 or pH 6-7 TEAP/ACN as flowing phase; and 360nm ultraviolet detection at column temperature 35deg c. The present invention has raised separation result reproducibility, optional water phase buffering area and trimmed pH value for analysis of chromatographic column and sample, and accurate analysis result. The present invention is suitable for common HPLC analysis lab and the application range includes various amino acid analysis, indirect analysis of other matter capable of being converted into amino acid, and other matter capable of taking quantitative deriving reaction with DNFB.
Description
Technical field
The present invention relates to amino acid whose analytical approach, based on phosphoric acid triethylamine/acetonitrile being the flowing opposite amino acid-2 that is separated, 4-dinitrofluorobenzene derivant, use common high performance liquid chromatograph and chromatographic condition, the instrument configuration that no longer needs other, just can finish the analysis of each seed amino acid sample, and provide result accurately.
Background technology
(common AA has kind more than 20 to amino acid for amino acid, AA) sample more complicated, and chromatogram analysis method must have enough selectivity, to differentiate AA to be determined in the sample.Most of AA do not have tangible absorption to ultraviolet light, and in order to detect, the most frequently used means are amino and the derivating agent reactions that make in the AA molecule, generate the derivant with uv absorption or photoluminescent property.
Usually adopt two kinds of path analysis AA samples.A kind of is to make AA separated from one another earlier by ion-exchange mechanism, detects with post-column derivation again.This is the employed way of general AA analyser.
Another approach is that AA is behind column front derivation, with the derivant of reversed phase chromatography separation AA.The column front derivation method is separated does not have special requirement to instrument and equipment, adopt the reverse phase separation pattern simultaneously, but require derivatization reaction can make every kind of AA almost entirely generate corresponding specific product, and there are enough detection sensitivities [Zhu Pengling, Yun Zihou, to thank to brilliance, " modern liquid chromatography ", the 22nd chapter, publishing house of Lanzhou University, 1989].
In the bibliographical information in early days, AA is through 2,4-dinitrofluorobenzene (DNFB) column front derivation, and reverse phase separation derivant AA-DNFB, separating DNFB-AA and be with 60mM sodium acetate (pH=4.45) is water, is organic phase with methyl alcohol, at C
18Carry out gradient elution [Morton R.C. on the post; Gerber G.E., Anal.Biochem., 170 (1988) 220.].Afterwards, the acetate buffer-acetonitrile system that uses pH=6.5-7 mostly was as moving phase, and making has enough degree of separation [Shang, Zhen-Hua between isoleucine and the leucine; Yu, Yi-Nian; Guo, Wei; Jiang, Hui; Zhou, Liang-Mo, Chinese Journal of Chemistry, 13 (1995) 163.].
Chinese patent CN 1053128A discloses the HPLC analytical method of 18 compound amino-acids injection liq, and it is with 2, and the 4-dinitrofluorobenzene is made derivative reagent, with pre-column derivatization method 18 compound amino-acids injection liq is carried out quantitative measurement.Detect liquid with quantitative dilution preparation, do the gradient elution agent with the moving phase acetonitrile of finite concentration and pH value and moving phase sodium acetate and acetate buffer solution.
Experiment shows that reverse phase separation AA-DNFB is very sensitive to the variation of moving phase pH value, and it is vital accurately controlling moving phase pH value.When using near the component of neutral acetate solution as moving phase, a tangible problem is that between the buffer zone of the acidity of moving phase away from this damping fluid, the durability of the method (robustness) just can not get guaranteeing.The not moving phase and the different brands of same date preparation, even the chromatographic column of the different lot numbers of same brand all may influence the chromatographic resolution result of DNFB-AA derivant.
Summary of the invention
The purpose of this invention is to provide a kind of amino acid whose analytical approach.With phosphoric acid triethylamine/acetonitrile is the flowing opposite amino acid-2 that is separated, and 4-dinitrofluorobenzene derivant can overcome the deficiency of prior art.The present invention replaces original acetate with phosphate, uses phosphoric acid triethylamine (TEAP)/acetonitrile (ACN) moving phase system, has improved the reappearance of separating resulting.Durability of the present invention is good, is easy to be adopted by common laboratory.By select between the water buffer zone and in interval little adjust pH, make analytical approach be applicable to different brands chromatographic column and various specimen types.Need not increase equipment, promptly can be general HPLC analysis room and adopt.
The amino acid whose analytical approach of the present invention comprises: use high performance liquid chromatograph, AA is through 2,4-dinitrofluorobenzene (DNFB) column front derivation, reverse phase separation derivant AA-DNFB, with phosphoric acid triethylamine/acetonitrile is eluent gradient wash-out or isocratic elution, reverse phase separation amino acid-2 on analytical column, 4-dinitrofluorobenzene derivant calculates amino acid whose content with the external standard method quantitative test again.
The amino acid whose analytical approach of the present invention comprises the steps:
1) prepares amino acid standard solution and sample solution with HCl solution as solvent respectively;
2) get standard solution and sample solution respectively in in the volumetric flask of 10 times of amounts of its volume, add 0.5MNaHCO
3, standard solution or sample solution and NaHCO
3Volume ratio be 1: 1; Add 1%2 again, the acetonitrile solution of 4-dinitrofluorobenzene, with the volume ratio of standard solution or sample solution be 0.5-1: 1, shake up; Place 60 ℃ of down heating 60 minutes, be cooled to room temperature, with 0.1M pH be 6.5 phosphate buffer towards to scale, shake up, standby;
3) use comprises the HPLC instrument that pump, mixer, constant temperature oven, UV-detector and workstation are formed.At C
18On the analytical column, with TEAP/ACN be moving phase, gradient elution or isocratic elution separate AA-DNFB; Chromatographic condition is as follows:
Analytical column C
18, 5 μ m, 250 * 4.6mm
Mobile phase A: ACN B:36mM TEAP, pH2-3 or 6-7
Type of elution gradient elution or isocratic elution
Detect UV300-380nm,
Temperature 20-45 ℃,
4) calculate with the external standard method quantitative test
The concentration of AA=(A in the sample
Sample/ A
Standard) * C
Standard
A
Sample: the peak area of AA in the sample chromatogram
A
Standard: the peak area of AA in the standard colors spectrogram
C
Standard: the concentration of standard solution AA.
Specific embodiments of the present invention comprises the steps:
1) prepares AA Standard solution and sample solution with 0.1M HCl as solvent respectively;
2) get 1mL standard solution and sample solution respectively in the brown volumetric flask of 10mL, add 1mL 0.5MNaHCO
3, shake up.Add the acetonitrile solution of 0.5-1mL 1%DNFB again, shake up; Place 60 ℃ of down heating 60 minutes, be cooled to room temperature, with 0.1M pH be 6.5 phosphate buffer towards to scale, shake up, standby.
3) use comprises the HPLC instrument that pump, mixer, constant temperature oven, UV-detector and workstation are formed.At C
18On the analytical column, with TEAP/ACN be moving phase, gradient elution or isocratic elution separate AA-DNFB.Chromatographic condition is as follows:
Analytical column C
18, 5 μ m, 250 * 4.6mm
Mobile phase A: ACN B:36mM TEAP, pH2-3 or 6-7
Type of elution gradient elution or isocratic elution
Detect UV300-380nm, preferred 360nm,
Temperature 20-45 ℃, preferred 35 ℃;
4) calculate with the external standard method quantitative test
The concentration of AA=(A in the sample
Sample/ A
Standard) * C
Standard
A
Sample: the peak area of AA in the sample chromatogram
A
Standard: the peak area of AA in the standard colors spectrogram
C
Standard: the concentration of standard solution AA.
The present invention has adopted phosphate to replace original acetate, has improved the durability of method.In this buffer system, the kation of triethylamine as phosphate radical to ion, rather than potassium or sodion.Between phosphatic buffer zone pH=2-3 and 6-8.In described AA analytical approach, with the 36mM phosphoric acid triethylamine, pH=2-3 and 6-7 aqueous solution/ACN moving phase system wash-out AA-DNFB (2, the 4-dinitrofluorobenzene).Secondly, the acidity of moving phase influence separation selectivity, is water with the TEAP of different pH, and the AA separation selectivity shows very big difference, and this provides convenience for the optimization chromatographic condition.In addition, the adding of triethylamine can suppress the influence of residual silicon hydroxyl to separating on the stationary phase.
The present invention uses phosphoric acid triethylamine (TEAP)/acetonitrile (ACN) moving phase, has improved the reappearance of separating resulting.By selecting between the water buffer zone and little adjust pH in interval, make analytical approach (for example: mensuration of principal ingredient etc. in parenteral solution class or the bulk drug) be applicable to different brands chromatographic column and various specimen types, solve the analysis of ispol, and provided result accurately.The present invention need not increase equipment, can be suitable for general HPLC analysis room and adopt.Range of application enlarges, and comprises the indirect analysis after various amino acid whose analyses and other material are converted into amino acid, comprises that also other can carry out the analysis of quantitative derivatization reaction material with DNFB except that amino acid.
Description of drawings
Fig. 1 is with 36mM TEAP, and pH6-7/ACN is a moving phase ephrosis parenteral solution chromatogram.
Fig. 2 is with 36mM TEAP, and pH6-7/ACN is a moving phase hepatopathy parenteral solution chromatogram.
Fig. 3 is with 36mM TEAP, and pH2-3/ACN is a moving phase ephrosis parenteral solution chromatogram.
Fig. 4 is with 36mM TEAP, and pH2-3/ACN is a moving phase hepatopathy parenteral solution chromatogram.
Fig. 5 is with 36mM TEAP, and pH2-3/ACN is a moving phase xylamidine acid hydrolysis solution chromatogram.
Fig. 6 is with 36mM TEAP, and pH2-3/ACN is a moving phase phenylalanine chromatogram.
Embodiment
Further specify the present invention below in conjunction with embodiment.
Embodiment 1 ephrosis parenteral solution (Amino acid for renal insufficiency,
Hoechst Marion Roussel company produces) and the hepatopathy parenteral solution (Amino acid preparation for hepaticinsufficiency,
Hoechst Marion Roussel company produces) in the analysis of AA
1. reagent
1.1.0.1M HCl 4mL hydrochloric acid mixes with the 480mL water.
1.2 1%DNFB 1g DNFB is dissolved in the 100mL acetonitrile.
1.3.0.5M NaHCO
32.1g NaHCO
3Be dissolved in the 50mL water.
1.4.0.1M phosphate buffer, pH6.5 claim 1.74g K
2HPO
4Be dissolved in and get 0.1MK in the 100mL water
2HPO
4Claim 1.36g KH
2PO
4Be dissolved in and get 0.1M KH in the 100mL water
2PO
4To 0.1M KH
2PO
4In add 0.1M K
2HPO
4To pH=6.5.
1.5.36mM phosphoric acid triethylamine (TEAP), pH2.75 are got in beaker～950mL water, get the 5mL triethylamine with transfer pipet and add to wherein.After the mixing, with phosphorus acid for adjusting pH value to 2.75.Solution is transferred in the 1000mL volumetric flask, and water dashes to scale.Filtering the back uses.
1.6.36mM phosphoric acid triethylamine (TEAP), pH6.50 are got in beaker～950mL water, get the 5mL triethylamine with transfer pipet and add to wherein.After the mixing, with phosphorus acid for adjusting pH value to 6.50.Solution is transferred in the 1000mL volumetric flask, and water dashes to scale.Filtering the back uses.
2. the preparation of standard solution accurately takes by weighing the AA object of reference by the composition of ephrosis parenteral solution and hepatopathy parenteral solution, makes the solvent constant volume with 0.1M HCl.Get AA Standard solution 5mL in the 25mL volumetric flask with transfer pipet, dash to scale, shake up standby with 0.1M HCl.
3. the preparation of sample solution is got AA injection liquid samples 1mL in the 25mL volumetric flask with transfer pipet, dashes to scale with 0.1M HCl, shakes up standby.
4. the derivatization reaction of standard solution and sample solution is got AA Standard solution and each 1mL of sample solution after the dilution with transfer pipet, places the brown volumetric flask of 10mL respectively, adds 1mL 0.5M NaHCO
3, shake up, add 1mL1%DNFB again, shake up.Place on 60 ℃ of heat blocks and heated 60 minutes.After the cooling, dash to scale, shake up, prepare HPLC and analyze usefulness with 0.1M phosphate buffer (pH6.5).
5. chromatographic condition
5.1. measure asparagus fern, paddy, dried meat, third, different bright, leucine
Analytical column Kromasil C
18, 5 μ m, 250 * 4.6mm
Mobile phase A: ACN B:36mM TEAP, pH6.50
Gradient 83/71/68/10/10B%[V
B/ (V
A+ V
B)] at 0/10/25/30/35min
Equilibration time 10min
Detect UV360nm
35 ℃ of temperature
Obtain the separating resulting as chromatogram 1 (ephrosis parenteral solution) and 2 (hepatopathy parenteral solutions) with this understanding, peak sequence is: asparagus fern, paddy, essence, silk, Soviet Union and sweet (with the reagent overlap of peaks), dried meat, third, figured silk fabrics, egg, different bright, bright, look, group, phenylpropyl alcohol, rely, tyrosine
5.2. measure essence, silk, Soviet Union, sweet, group, egg, figured silk fabrics, look, phenylpropyl alcohol, bad, tyrosine
Analytical column Kromasil C
18, 5 μ m, 250 * 4.6mm
Mobile phase A: ACN B:36mM TEAP, pH2.75
Gradient 78/60/35/10/10B%[V
B/ (V
A+ V
B)] at 0/20/30/35/40min
Equilibration time 10min
Detect UV360nm
35 ℃ of temperature
Obtain the separating resulting as chromatogram 3 (ephrosis parenteral solution) and 4 (hepatopathy parenteral solutions) with this understanding, peak sequence is: essence, silk, asparagus fern, paddy, Soviet Union, sweet, third, dried meat, group, egg, figured silk fabrics, look, phenylpropyl alcohol, bright+different bright, bad, tyrosine.
6. calculate
The concentration of AA=(A in the sample
Sample/ A
Standard) * C
Standard
A
Sample: the peak area of AA in the sample chromatogram
A
Standard: the peak area of AA in the standard colors spectrogram
C
Standard: the concentration of standard solution AA
7. the analysis result of ephrosis parenteral solution, accuracy, precision
Here be example with the ephrosis parenteral solution, provide the accuracy and the precision of method.The hepatopathy parenteral solution also provides close result.
7.1. accuracy:
Table 1 accuracy determination result (%RSD, relative standard deviation)
| Aspartic acid | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | ％RSD |
| 1 | Response | 107505 | 159941 | 212435 | 264899 | 317737 | |
| The linearity correction measured value | 0.5004 | 0.7499 | 0.9997 | 1.2493 | 1.5007 | ||
| The recovery 100% | 99.99 | 99.97 | 99.94 | 0.02 | |||
| 2 | Response | 107609 | 159240 | 212106 | 265468 | 316251 | |
| The linearity correction measured value | 0.5009 | 0.7474 | 0.9999 | 1.2547 | 1.4972 | ||
| The recovery 100% | 99.66 | 99.99 | 100.37 | 0.36 | |||
| 3 | Response | 106905 | 160295 | 212177 | 265850 | 315048 | |
| The linearity correction measured value | 0.4964 | 0.7521 | 1.0006 | 1.2577 | 1.4933 | ||
| The recovery 100% | 100.28 | 100.06 | 100.61 | 0.28 | |||
| Glutamic acid | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 82767 | 125208 | 169925 | 214174 | 262019 |
| The linearity correction measured value | 0.5083 | 0.7453 | 0.9950 | 1.2421 | 1.5093 | ||
| The recovery 100% | 99.37 | 99.50 | 99.37 | 0.08 | |||
| 2 | Response | 81268 | 123844 | 167062 | 211833 | 257995 | |
| The linearity correction measured value | 0.5067 | 0.7477 | 0.9924 | 1.2459 | 1.5073 | ||
| The recovery 100% | 99.70 | 99.24 | 99.67 | 0.26 | |||
| 3 | Response | 81949 | 124875 | 169259 | 214294 | 258174 | |
| The linearity correction measured value | 0.5035 | 0.7463 | 0.9974 | 1.2522 | 1.5005 | ||
| The recovery 100% | 99.51 | 99.74 | 100.18 | 0.34 | |||
| Proline | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 674060 | 1016544 | 1349867 | 1695593 | 2030976 | |
| The linearity correction measured value | 0.4994 | 0.7518 | 0.9974 | 1.2521 | 1.4992 | ||
| The recovery 100% | 100.24 | 99.74 | 100.17 | 0.27 | |||
| 2 | Response | 671004 | 1011913 | 1349929 | 1699227 | 2026416 | |
| The linearity correction measured value | 0.4993 | 0.7500 | 0.9987 | 1.2557 | 1.4964 | ||
| The recovery 100% | 100.01 | 99.87 | 100.45 | 0.30 | |||
| 3 | Response | 677057 | 1013637 | 1358996 | 1694995 | 2037505 | |
| The linearity correction measured value | 0.5008 | 0.7481 | 1.0019 | 1.2488 | 1.5004 | ||
| The recovery 100% | 99.75 | 100.19 | 99.90 | 0.22 | |||
| Alanine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 1689226 | 2529193 | 3358129 | 4213693 | 5039937 | |
| The linearity correction measured value | 0.5001 | 0.7505 | 0.9976 | 1.2527 | 1.4990 | ||
| The recovery 100% | 100.07 | 99.76 | 100.22 | 0.23 | |||
| 2 | Response | 1684704 | 2526283 | 3357122 | 4207109 | 5048707 | |
| The linearity correction measured value | 0.5005 | 0.7507 | 0.9977 | 1.2504 | 1.5006 | ||
| The recovery 100% | 100.09 | 99.77 | 100.03 | 0.17 | |||
| 3 | Response | 1686922 | 2530517 | 3370821 | 4203437 | 5059251 | |
| The linearity correction measured value | 0.5001 | 0.7506 | 1.0002 | 1.2475 | 1.5016 | ||
| The recovery 100% | 100.08 | 100.02 | 99.80 | 0.15 | |||
| Isoleucine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 2847226 | 4278219 | 5702401 | 7146538 | 8590133 | |
| The linearity correction measured value | 0.5009 | 0.7501 | 0.9982 | 1.2497 | 1.5011 | ||
| The recovery 100% | 100.02 | 99.82 | 99.98 | 0.11 | |||
| 2 | Response | 2846123 | 4271169 | 5711365 | 7156548 | 8589588 | |
| The linearity correction measured value | 0.5010 | 0.7489 | 0.9994 | 1.2508 | 1.5000 | ||
| The recovery 100% | 99.85 | 99.94 | 100.06 | 0.11 | |||
| 3 | Response | 2846518 | 4279660 | 5713699 | 7160764 | 8601874 | |
| The linearity correction measured value | 0.5008 | 0.7497 | 0.9988 | 1.2502 | 1.5005 | ||
| The recovery 100% | 99.96 | 99.88 | 100.01 | 0.07 | |||
| Leucine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 3877328 | 5830216 | 7766820 | 9760839 | 11695672 | |
| The linearity correction measured value | 0.5006 | 0.7501 | 0.9975 | 1.2523 | 1.4995 | ||
| The recovery 100% | 100.01 | 99.75 | 100.18 | 0.22 | |||
| 2 | Response | 3871704 | 5822899 | 7779548 | 9744576 | 11704465 | |
| The linearity correction measured value | 0.5006 | 0.7496 | 0.9994 | 1.2502 | 1.5003 |
| The recovery 100% | 99.95 | 99.94 | 100.01 | 0.04 | |||
| 3 | Response | 3885949 | 5847103 | 7779066 | 9760675 | 11707404 | |
| The linearity correction measured value | 0.5002 | 0.7509 | 0.9978 | 1.2511 | 1.5000 | ||
| The recovery 100% | 100.11 | 99.78 | 100.09 | 0.19 | |||
| Arginine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 835750 | 1242453 | 1661765 | 2095025 | 2511935 | |
| The linearity correction measured value | 0.5044 | 0.7462 | 0.9955 | 1.2530 | 1.5009 | ||
| The recovery 100% | 99.49 | 99.55 | 100.24 | 0.42 | |||
| 2 | Response | 826133 | 1244503 | 1649347 | 2084369 | 2492214 | |
| The linearity correction measured value | 0.5008 | 0.7515 | 0.9940 | 1.2547 | 1.4990 | ||
| The recovery 100% | 100.19 | 99.40 | 100.37 | 0.52 | |||
| 3 | Response | 829654 | 1249113 | 1660278 | 2094206 | 2485311 | |
| The linearity correction measured value | 0.4984 | 0.7507 | 0.9979 | 1.2589 | 1.4941 | ||
| The recovery 100% | 100.09 | 99.79 | 100.71 | 0.47 | |||
| Serine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 461403 | 697391 | 927173 | 1171787 | 1408063 | |
| The linearity correction measured value | 0.5019 | 0.7511 | 0.9937 | 1.2519 | 1.5014 | ||
| The recovery 100% | 100.14 | 99.37 | 100.15 | 0.45 | |||
| 2 | Response | 456751 | 691675 | 924844 | 1167655 | 1395277 | |
| The linearity correction measured value | 0.5002 | 0.7497 | 0.9975 | 1.2554 | 1.4972 | ||
| The recovery 100% | 99.97 | 99.75 | 100.43 | 0.35 | |||
| 3 | Response | 462885 | 699799 | 935278 | 1170366 | 1397845 | |
| The linearity correction measured value | 0.4976 | 0.7507 | 1.0022 | 1.2533 | 1.4962 | ||
| The recovery 100% | 100.09 | 100.22 | 100.26 | 0.09 | |||
| Threonine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 970437 | 1462010 | 1942883 | 2437173 | 2921780 | |
| The linearity correction measured value | 0.4996 | 0.7515 | 0.9980 | 1.2513 | 1.4997 | ||
| The recovery 100% | 100.20 | 99.80 | 100.10 | 0.21 | |||
| 2 | Response | 969140 | 1458548 | 1942414 | 2443789 | 2923002 | |
| The linearity correction measured value | 0.5002 | 0.7502 | 0.9975 | 1.2536 | 1.4985 | ||
| The recovery 100% | 100.03 | 99.75 | 100.29 | 0.27 | |||
| 3 | Response | 969102 | 1458788 | 1945405 | 2438242 | 2925560 | |
| The linearity correction measured value | 0.5001 | 0.7503 | 0.9990 | 1.2508 | 1.4998 | ||
| The recovery 100% | 100.04 | 99.90 | 100.06 | 0.09 | |||
| Glycocoll | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 894958 | 1350889 | 1807642 | 2289228 | 2745832 | |
| The linearity correction measured value | 0.5029 | 0.7485 | 0.9946 | 1.2540 | 1.5000 | ||
| The recovery 100% | 99.80 | 99.46 | 100.32 | 0.44 | |||
| 2 | Response | 890634 | 1325487 | 1776681 | 2236567 | 2671139 | |
| The linearity correction measured value | 0.5028 | 0.7459 | 0.9981 | 1.2552 | 1.4981 | ||
| The recovery 100% | 99.45 | 99.81 | 100.41 | 0.49 | |||
| 3 | Response | 895637 | 1325477 | 1787844 | 2246018 | 2669270 | |
| The linearity correction measured value | 0.5026 | 0.7430 | 1.0017 | 1.2580 | 1.4948 | ||
| The recovery 100% | 99.07 | 100.17 | 100.64 | 0.81 |
| Histidine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 775169 | 1155863 | 1521847 | 1888645 | 2208544 | |
| The linearity correction measured value | 0.4901 | 0.7543 | 1.0082 | 1.2627 | 1.4847 | ||
| The recovery 100% | 100.57 | 100.82 | 101.02 | 0.22 | |||
| 2 | Response | 769331 | 1131464 | 1510620 | 1871636 | 2227754 | |
| The linearity correction measured value | 0.4991 | 0.7466 | 1.0058 | 1.2526 | 1.4960 | ||
| The recovery 100% | 99.55 | 100.58 | 100.20 | 0.52 | |||
| 3 | Response | 765605 | 1139491 | 1492895 | 1838605 | 2158399 | |
| The linearity correction measured value | 0.4886 | 0.7566 | 1.0100 | 1.2578 | 1.4870 | ||
| The recovery 100% | 100.88 | 101.00 | 100.62 | 0.19 | |||
| Methionine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 1541638 | 2315268 | 3078507 | 3828190 | 4612621 | |
| The linearity correction measured value | 0.4992 | 0.7518 | 1.0011 | 1.2459 | 1.5021 | ||
| The recovery 100% | 100.24 | 100.11 | 99.67 | 0.30 | |||
| 2 | Response | 1525963 | 2280448 | 3039779 | 3800354 | 4568568 | |
| The linearity correction measured value | 0.5013 | 0.7493 | 0.9989 | 1.2490 | 1.5015 | ||
| The recovery 100% | 99.91 | 99.89 | 99.92 | 0.01 | |||
| 3 | Response | 1535531 | 2285886 | 3041390 | 3828625 | 4575687 | |
| The linearity correction measured value | 0.5022 | 0.7483 | 0.9961 | 1.2542 | 1.4992 | ||
| The recovery 100% | 99.77 | 99.61 | 100.34 | 0.38 | |||
| Valine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 2870482 | 4302568 | 5634504 | 7063143 | 8408206 | |
| The linearity correction measured value | 0.4968 | 0.7555 | 0.9962 | 1.2543 | 1.4973 | ||
| The recovery 100% | 100.74 | 99.62 | 100.34 | 0.57 | |||
| 2 | Response | 2827334 | 4244625 | 5665077 | 7035612 | 8393130 | |
| The linearity correction measured value | 0.4962 | 0.7507 | 1.0057 | 1.2518 | 1.4955 | ||
| The recovery 100% | 100.09 | 100.57 | 100.14 | 0.26 | |||
| 3 | Response | 2841364 | 4242346 | 5644377 | 7083558 | 8427622 | |
| The linearity correction measured value | 0.4994 | 0.7493 | 0.9994 | 1.2561 | 1.4959 | ||
| The recovery 100% | 99.90 | 99.94 | 100.49 | 0.33 | |||
| Tryptophane | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 556880 | 847486 | 1131498 | 1409325 | 1712273 | |
| The linearity correction measured value | 0.5000 | 0.7529 | 1.0000 | 1.2418 | 1.5054 | ||
| The recovery 100% | 100.38 | 100.00 | 99.34 | 0.53 | |||
| 2 | Response | 555125 | 827557 | 1111071 | 1398090 | 1689129 | |
| The linearity correction measured value | 0.5059 | 0.7458 | 0.9955 | 1.2482 | 1.5045 | ||
| The recovery 100% | 99.44 | 99.55 | 99.86 | 0.22 | |||
| 3 | Response | 553313 | 838300 | 1122892 | 1403467 | 1683358 | |
| The linearity correction measured value | 0.4983 | 0.7505 | 1.0023 | 1.2506 | 1.4983 | ||
| The recovery 100% | 100.07 | 100.23 | 100.05 | 0.10 | |||
| Phenylalanine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 1393886 | 2095567 | 2758770 | 3465822 | 4166411 | |
| The linearity correction measured value | 0.5004 | 0.7540 | 0.9937 | 1.2493 | 1.5026 |
| The recovery 100% | 100.53 | 99.37 | 99.95 | 0.58 | |||
| 2 | Response | 1374051 | 2064323 | 2750768 | 3438517 | 4139278 | |
| The linearity correction measured value | 0.5006 | 0.7505 | 0.9991 | 1.2481 | 1.5018 | ||
| The recovery 100% | 100.07 | 99.91 | 99.85 | 0.12 | |||
| 3 | Response | 1381206 | 2077333 | 2745871 | 3440346 | 4117863 | |
| The linearity correction measured value | 0.4985 | 0.7531 | 0.9976 | 1.2515 | 1.4993 | ||
| The recovery 100% | 100.41 | 99.76 | 100.12 | 0.33 | |||
| Lysine | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | |
| 1 | Response | 3009342 | 4493168 | 5934873 | 7335943 | 8679634 | |
| The linearity correction measured value | 0.4923 | 0.7538 | 1.0078 | 1.2547 | 1.4914 | ||
| The recovery 100% | 100.50 | 100.78 | 100.37 | 0.21 | |||
| 2 | Response | 3016029 | 4473573 | 5928032 | 7343220 | 8683054 | |
| The linearity correction measured value | 0.4945 | 0.7510 | 1.0069 | 1.2559 | 1.4917 | ||
| The recovery 100% | 100.13 | 100.69 | 100.47 | 0.28 | |||
| 3 | Response | 3012738 | 4500359 | 5927950 | 7328485 | 8678348 | |
| The linearity correction measured value | 0.4922 | 0.7548 | 1.0068 | 1.2540 | 1.4922 | ||
| The recovery 100% | 100.64 | 100.68 | 100.32 | 0.20 | |||
| Tyrosine | 50％ | 75％ | 100％ | 125％ | 150％ | ||
| 1 | Response | 132634 | 198614 | 263986 | 329785 | 398749 | |
| The linearity correction measured value | 0.5022 | 0.7508 | 0.9971 | 1.2450 | 1.5049 | ||
| The recovery 100% | 100.10 | 99.71 | 99.60 | 0.26 | |||
| 2 | Response | 131624 | 195033 | 262815 | 329784 | 400745 | |
| The linearity correction measured value | 0.5084 | 0.7439 | 0.9956 | 1.2443 | 1.5078 | ||
| The recovery 100% | 99.19 | 99.56 | 99.54 | 0.21 | |||
| 3 | Response | 131952 | 196135 | 264876 | 331776 | 401083 | |
| The linearity correction measured value | 0.5059 | 0.7440 | 0.9989 | 1.2471 | 1.5041 | ||
| The recovery 100% | 99.19 | 99.89 | 99.77 | 0.37 |
To the AA of all tests, the recovery is all in 100 ± 1% scopes.
7.2. precision:
This method has higher precision, and middle precision and repeatability all are not more than 0.5%, below is measurement result.
7.2.1. middle precision:
Table 2: middle precision measurement result
| Aspartic acid | Concentration | 50％ | 75％ | 100％ | 125％ | 150％ | Mean value |
| Measured value | 0.5004 | 0.7499 | 0.9997 | 1.2493 | 1.5007 | ||
| Measured value | 0.5009 | 0.7474 | 0.9999 | 1.2547 | 1.4972 | ||
| Measured value | 0.4964 | 0.7521 | 1.0006 | 1.2577 | 1.4933 | ||
| SD | 2.5E-03 | 2.3E-03 | 4.8E-04 | 4.2E-03 | 3.7E-03 | ||
| ％RSD | 0.50 | 0.31 | 0.05 | 0.34 | 0.25 | 0.29 | |
| Glutamic acid | Measured value | 0.5083 | 0.7453 | 0.9950 | 1.2421 | 1.5093 | |
| Measured value | 0.5067 | 0.7477 | 0.9924 | 1.2459 | 1.5073 | ||
| Measured value | 0.5035 | 0.7463 | 0.9974 | 1.2522 | 1.5005 | ||
| SD | 2.4E-03 | 1.2E-03 | 2.5E-03 | 5.1E-03 | 4.6E-03 | ||
| ％RSD | 0.49 | 0.16 | 0.25 | 0.41 | 0.31 | 0.32 |
| Proline | Measured value 1 | 0.4994 | 0.7518 | 0.9974 | 1.2521 | 1.4992 | |
| Measured value 2 | 0.4993 | 0.7500 | 0.9987 | 1.2557 | 1.4964 | ||
| Measured value 3 | 0.5008 | 0.7481 | 1.0019 | 1.2488 | 1.5004 | ||
| SD | 8.4E-04 | 1.8E-03 | 2.3E-03 | 3.4E-03 | 2.1E-03 | ||
| ％RSD | 0.17 | 0.25 | 0.23 | 0.28 | 0.14 | 0.21 | |
| Alanine | Measured value 1 | 0.5001 | 0.7505 | 0.9976 | 1.2527 | 1.4990 | |
| Measured value 2 | 0.5005 | 0.7507 | 0.9977 | 1.2504 | 1.5006 | ||
| Measured value 3 | 0.5001 | 0.7506 | 1.0002 | 1.2475 | 1.5016 | ||
| SD | 2.3E-04 | 9.2E-05 | 1.4E-03 | 2.6E-03 | 1.3E-03 | ||
| ％RSD | 0.05 | 0.01 | 0.14 | 0.21 | 0.09 | 0.10 | |
| Isoleucine | Measured value 1 | 0.5009 | 0.7501 | 0.9982 | 1.2497 | 1.5011 | |
| Measured value 2 | 0.5010 | 0.7489 | 0.9994 | 1.2508 | 1.5000 | ||
| Measured value 3 | 0.5008 | 0.7497 | 0.9988 | 1.2502 | 1.5005 | ||
| SD | 1.1E-04 | 6.5E-04 | 6.0E-04 | 5.3E-04 | 5.4E-04 | ||
| ％RSD | 0.02 | 0.09 | 0.06 | 0.04 | 0.04 | 0.05 | |
| Leucine | Measured value 1 | 0.5006 | 0.7501 | 0.9975 | 1.2523 | 1.4995 | |
| Measured value 2 | 0.5006 | 0.7496 | 0.9994 | 1.2502 | 1.5003 | ||
| Measured value 3 | 0.5002 | 0.7509 | 0.9978 | 1.2511 | 1.5000 | ||
| SD | 2.5E-04 | 6.3E-04 | 9.8E-04 | 1.1E-03 | 4.1E-04 | ||
| ％RSD | 0.05 | 0.08 | 0.10 | 0.09 | 0.03 | 0.07 | |
| Arginine | Measured value 1 | 0.5044 | 0.7462 | 0.9955 | 1.2530 | 1.5009 | |
| Measured value 2 | 0.5008 | 0.7515 | 0.9940 | 1.2547 | 1.4990 | ||
| Measured value 3 | 0.4984 | 0.7507 | 0.9979 | 1.2589 | 1.4941 | ||
| SD | 3.0E-03 | 2.8E-03 | 2.0E-03 | 3.0E-03 | 3.5E-03 | ||
| ％RSD | 0.60 | 0.38 | 0.20 | 0.24 | 0.23 | 0.33 | |
| Serine | Measured value 1 | 0.5019 | 0.7511 | 0.9937 | 1.2519 | 1.5014 | |
| Measured value 2 | 0.5002 | 0.7497 | 0.9975 | 1.2554 | 1.4972 | ||
| Measured value 3 | 0.4976 | 0.7507 | 1.0022 | 1.2533 | 1.4962 | ||
| SD | 2.2E-03 | 6.9E-04 | 4.3E-03 | 1.8E-03 | 2.7E-03 | ||
| ％RSD | 0.43 | 0.09 | 0.43 | 0.14 | 0.18 | 0.25 | |
| Threonine | Measured value 1 | 0.4996 | 0.7515 | 0.9980 | 1.2513 | 1.4997 | |
| Measured value 2 | 0.5002 | 0.7502 | 0.9975 | 1.2536 | 1.4985 | ||
| Measured value 3 | 0.5001 | 0.7503 | 0.9990 | 1.2508 | 1.4998 | ||
| SD | 3.3E-04 | 7.1E-04 | 7.7E-04 | 1.5E-03 | 7.4E-04 | ||
| ％RSD | 0.07 | 0.09 | 0.08 | 0.12 | 0.05 | 0.08 | |
| Glycocoll | Measured value 1 | 0.5029 | 0.7485 | 0.9946 | 1.2540 | 1.5000 | |
| Measured value 2 | 0.5028 | 0.7459 | 0.9981 | 1.2552 | 1.4981 | ||
| Measured value 3 | 0.5026 | 0.7430 | 1.0017 | 1.2580 | 1.4948 | ||
| SD | 1.7E-04 | 2.7E-03 | 3.5E-03 | 2.0E-03 | 2.7E-03 | ||
| ％RSD | 0.03 | 0.37 | 0.35 | 0.16 | 0.18 | 0.22 | |
| Histidine | Measured value 1 | 0.4901 | 0.7543 | 1.0082 | 1.2627 | 1.4847 | |
| Measured value 2 | 0.4991 | 0.7466 | 1.0058 | 1.2526 | 1.4960 | ||
| Measured value 3 | 0.4886 | 0.7566 | 1.0100 | 1.2578 | 1.4870 |
| SD | 5.7E-03 | 5.2E-03 | 2.1E-03 | 5.1E-03 | 6.0E-03 | ||
| ％RSD | 1.13 | 0.70 | 0.21 | 0.41 | 0.40 | 0.57 | |
| Methionine | Measured value 1 | 0.4992 | 0.7518 | 1.0011 | 1.2459 | 1.5021 | |
| Measured value 2 | 0.5013 | 0.7493 | 0.9989 | 1.2490 | 1.5015 | ||
| Measured value 3 | 0.5022 | 0.7483 | 0.9961 | 1.2542 | 1.4992 | ||
| SD | 1.6E-03 | 1.8E-03 | 2.5E-03 | 4.2E-03 | 1.5E-03 | ||
| ％RSD | 0.32 | 0.24 | 0.25 | 0.34 | 0.10 | 0.25 | |
| Valine | Measured value 1 | 0.4968 | 0.7555 | 0.9962 | 1.2543 | 1.4973 | |
| Measured value 2 | 0.4962 | 0.7507 | 1.0057 | 1.2518 | 1.4955 | ||
| Measured value 3 | 0.4994 | 0.7493 | 0.9994 | 1.2561 | 1.4959 | ||
| SD | 1.7E-03 | 3.3E-03 | 4.9E-03 | 2.2E-03 | 9.2E-04 | ||
| ％RSD | 0.34 | 0.44 | 0.49 | 0.17 | 0.06 | 0.30 | |
| Tryptophane | Measured value 1 | 0.5000 | 0.7529 | 1.0000 | 1.2418 | 1.5054 | |
| Measured value 2 | 0.5059 | 0.7458 | 0.9955 | 1.2482 | 1.5045 | ||
| Measured value 3 | 0.4983 | 0.7505 | 1.0023 | 1.2506 | 1.4983 | ||
| SD | 4.0E-03 | 3.6E-03 | 3.5E-03 | 4.6E-03 | 3.9E-03 | ||
| ％RSD | 0.80 | 0.48 | 0.35 | 0.37 | 0.26 | 0.45 | |
| Phenylalanine | Measured value 1 | 0.5004 | 0.7540 | 0.9937 | 1.2493 | 1.5026 | |
| Measured value 2 | 0.5006 | 0.7505 | 0.9991 | 1.2481 | 1.5018 | ||
| Measured value 3 | 0.4985 | 0.7531 | 0.9976 | 1.2515 | 1.4993 | ||
| SD | 1.1E-03 | 1.8E-03 | 2.7E-03 | 1.8E-03 | 1.7E-03 | ||
| ％RSD | 0.22 | 0.24 | 0.27 | 0.14 | 0.11 | 0.20 | |
| Lysine | Measured value 1 | 0.4923 | 0.7538 | 1.0078 | 1.2547 | 1.4914 | |
| Measured value 2 | 0.4945 | 0.7510 | 1.0069 | 1.2559 | 1.4917 | ||
| Measured value 3 | 0.4922 | 0.7548 | 1.0068 | 1.2540 | 1.4922 | ||
| SD | 1.3E-03 | 2.0E-03 | 5.6E-04 | 9.9E-04 | 4.1E-04 | ||
| ％RSD | 0.26 | 0.26 | 0.06 | 0.08 | 0.03 | 0.14 | |
| Tyrosine | Measured value 1 | 0.5022 | 0.7508 | 0.9971 | 1.2450 | 1.5049 | |
| Measured value 2 | 0.5084 | 0.7439 | 0.9956 | 1.2443 | 1.5078 | ||
| Measured value 3 | 0.5059 | 0.7440 | 0.9989 | 1.2471 | 1.5041 | ||
| SD | 3.2E-03 | 4.0E-03 | 1.7E-03 | 1.4E-03 | 1.9E-03 | ||
| ％RSD | 0.63 | 0.53 | 0.17 | 0.12 | 0.13 | 0.31 |
7.2.2. repeatability:
Table 3: repeated measurement result (SD standard deviation)
| AA | Six parts of 100%AA liquor sample responses | Mean value | SD | ％RS D | |||||
| 1 | 2 | 3 | 4 | 5 | 6 | ||||
| Aspartic acid | 282213 | 282838 | 281975 | 282412 | 283246 | 283707 | 282731.8 | 658.9 | 0.23 |
| Glutamic acid | 114212 | 113094 | 112932 | 113514 | 113567 | 112901 | 113370 | 501.3 | 0.44 |
| Proline | 1511838 | 1515825 | 1506534 | 1506317 | 1513907 | 1509467 | 1510648 | 3896.6 | 0.26 |
| Alanine | 3318444 | 3322253 | 3308888 | 3307867 | 3319691 | 3313502 | 3315108 | 5947.8 | 0.18 |
| Isoleucine | 5865799 | 5864773 | 5862861 | 5857414 | 5858293 | 5864464 | 5862267 | 3557.5 | 0.06 |
| Leucine | 8026932 | 8025250 | 8022505 | 8019344 | 8018902 | 8025567 | 8023083 | 3390.5 | 0.04 |
| Arginine | 1744130 | 1725521 | 1727469 | 1728244 | 1730010 | 1732531 | 1731318 | 6711.2 | 0.39 |
| Serine | 951103 | 949570 | 951600 | 945253 | 952324 | 950975 | 950137.5 | 2558.7 | 0.27 |
| Threonine | 1866838 | 1861626 | 1864604 | 1859593 | 1864654 | 1863245 | 1863427 | 2549.6 | 0.14 |
| Glycocoll | 1780536 | 1775098 | 1789397 | 1775533 | 1794689 | 1783383 | 1783106 | 7770.5 | 0.44 |
| Histidine | 1512464 | 1510513 | 1523605 | 1507769 | 1505902 | 1520203 | 1513409 | 7036.5 | 0.46 |
| Methionine | 3226137 | 3225164 | 3228301 | 3220109 | 3226790 | 3229103 | 3225934 | 3191.9 | 0.10 |
| Valine | 4997805 | 5001130 | 4996398 | 4992264 | 4999446 | 5000763 | 4997968 | 3317.3 | 0.07 |
| Tryptophane | 1033196 | 1026753 | 1036733 | 1036675 | 1033353 | 1035042 | 1033625 | 3700.1 | 0.36 |
| Phenylalanine | 2703400 | 2706028 | 2709602 | 2701132 | 2697518 | 2714989 | 2705445 | 6234.3 | 0.23 |
| Lysine | 5839359 | 5823697 | 5821899 | 5875818 | 5821530 | 5810851 | 5832192 | 23245.7 | 0.40 |
| Tyrosine | 238750 | 237775 | 238836 | 239670 | 238830 | 238934 | 238799.2 | 605.0 | 0.25 |
Major component xylamidine Determination on content in embodiment 2 xylamidines (L-alanyl-L-glutamine) bulk drug (Tianjin Tiancheng Pharmaceutical Co., Ltd.'s production)
1. reagent
1.1. referring to embodiment 1.
1.2.6N HCl concentrated hydrochloric acid and water mix by 1: 1 (V/V).
2. the xylamidine primary standard substance is taken out in the preparation of standard solution from exsiccator, accurately takes by weighing about 15mg in polyethylene pipe with cover (1), adds 1mL 6N HCl with transfer pipet, covers completely, shakes up standby.
3. the preparation of sample solution accurately takes by weighing the about 15mg of xylamidine sample in polyethylene pipe with cover (2), adds 1mL 6N HCl with transfer pipet, covers completely, shakes up standby.
4. the acidolysis of standard solution and sample solution is little in 105 ℃ of heating 6 with polyethylene pipe (1) and (2).Cooling shakes up standby.
5. the preparation without the acidolysis sample solution accurately takes by weighing the about 15mg of xylamidine sample in polyethylene pipe with cover (3), adds 1mL water with transfer pipet, covers completely, shakes up standby.
6. derivatization reaction respectively from polyethylene pipe (1), (2) and (3) each 20L of draw solution, places the brown volumetric flask of 10mL with transfer pipet.Add 1mL 0.5M NaHCO respectively
3, shake up.Add 0.5mL 1%DNFB again, shake up.Solution is placed heat block, and 60 ℃ were heated 60 minutes, took out cooling.Dash to scale with 0.1M phosphate buffer (pH6.5), shake up, prepare HPLC and analyze usefulness.
7. chromatographic condition
Analytical column Kromasil C
18, 5 μ m, 250 * 4.6mm
Mobile phase A: ACN B:36mM TEAP, pH2.75
Gradient 78/64.5/60/60B%[V
B/ (V
A+ V
B)] at0/15/25/30min
Equilibration time 10min
Detect UV360nm
35 ℃ of temperature
Obtain separating resulting with this understanding as chromatogram 5.
8. calculate
8.1. when the peak area of Ala-Glu, L-Glu derivant in the acidolysis sample spectrogram not less than Ala-Gln derivative peak area 0.1% the time, just do not consider the fixed influence of these impurity Dui Measuring.
The percentage composition of xylamidine=[(A
Sample:* W
Standard)/(A
Standard* W
Sample)] * 100%
W
Sample: acidolysis samples weighed (mg)
W
Standard: xylamidine primary standard substance heavy (mg)
A
Sample: glutamate derivatives peak area in the acidolysis sample spectrogram
A
Standard: glutamate derivatives peak area in the primary standard substance spectrogram
8.2. when the peak area of Ala-Glu, L-Glu in the acidolysis sample spectrogram not greater than the Ala-Gln peak area 0.1% the time, the measurement result that should proofread and correct xylamidine.Xylamidine is the degradation experiment result in the presence of acid, alkali, oxygenant show, Ala-Glu is main degradation product, but do not observe L-Glu.Therefore, timing is mainly considered original Ala-Glu in the sample.
The percentage composition of xylamidine={ [A
Sample* A
Ala-Gln* W
Standard]/[(A
Ala-Gln+ A
Ala-Glu* f) * (A
Standard* W
Sample)] * 100%
W
Sample: acidolysis samples weighed (mg)
W
Standard: xylamidine primary standard substance heavy (mg)
A
Sample: glutamate derivatives peak area in the acidolysis sample spectrogram
A
Standard: glutamate derivatives peak area in the primary standard substance spectrogram
A
Ala-Gln:: the peak area of xylamidine derivant in the acidolysis sample spectrogram not
A
Ala-Glu:: the peak area of glutamine dipeptide derivant in the acidolysis sample spectrogram not
F: xylamidine and glutamine dipeptide relative response factor.
Xylamidine Determination on content in the embodiment 3 xylamidine parenteral solutions (Kelun Pharm Ind Co., Ltd., Sichuan's production)
1. reagent
1.1. referring to embodiment 1.
1.2.6.7N HCl 167mL concentrated hydrochloric acid is diluted with water to 300mL.
2. the preparation of standard solution accurately takes by weighing about 20mg xylamidine or 13.5mg glutamic acid primary standard substance in polyethylene pipe with cover (1), adds 100 μ L water and 900 μ L 6.7N HCl, covers completely, shakes up standby.
3. the preparation of sample solution is got 100 μ L sample solutions and 900 μ L 6.7N HCl in polyethylene pipe with cover (2), covers completely, shakes up standby.
4. acidolysis places heat block with polyethylene pipe (1) and (2), and 105 ℃ of heating were taken out cooling after 6 hours, shook up standby.
5. derivatization reaction is drawn acidolysis standard solution and each 20mL of sample solution respectively in the brown volumetric flask of 10mL with transfer pipet.Add 1mL 0.5M NaHCO respectively
3, shake up.Add 0.5mL 1%DNFB again, shake up.Volumetric flask is placed heat block, and 60 ℃ were heated 60 minutes, took out cooling.Dash to scale with 0.1M phosphate buffer (pH6.5), shake up, prepare HPLC and analyze usefulness.
6. chromatographic condition
Analytical column Kromasil C
18, 5 μ m, 250 * 4.6mm
Mobile phase A: ACN B:36mM TEAP, pH2.75
Gradient 78/64.5/60/60B%[V
B/ (V
A+ V
B)] at0/15/25/30min
Equilibration time 10min
Detect UV360nm
35 ℃ of temperature
Obtain separating resulting with this understanding as chromatogram 5.
7. calculate
7.1. when being primary standard substance with the xylamidine, the concentration of xylamidine is pressed examination and is calculated:
The concentration of xylamidine (g/100mL)=A
Sample* C
Standard/ A
Standard
C
Standard: xylamidine primary standard substance solution concentration is (with primary standard substance weightometer in the 100L solution
Calculate, g/100mL)
A
Sample: glutamate derivatives peak area in the sample spectrogram
A
Standard: glutamate derivatives peak area in the primary standard substance spectrogram
7.2. when being primary standard substance with glutamic acid, the concentration of xylamidine is pressed examination and is calculated:
The concentration of xylamidine (g/100mL)=(A
Sample* C
Standard/ A
Standard) * (M
Ala-Gln/ M
L-Glu)
C
Standard: xylamidine primary standard substance solution concentration is (with primary standard substance weightometer in the 100L solution
Calculate, g/100mL)
A
Sample: glutamate derivatives peak area in the sample spectrogram
A
Standard: glutamate derivatives peak area in the primary standard substance spectrogram
M
Ala-Gln: the xylamidine molar weight
M
L-Glu: the glutamic acid molar weight.
The mensuration of major component phenylalanine content in the embodiment 4 L-phenylalanine bulk drugs
1. reagent is referring to embodiment 1.
2. the phenylalanine primary standard substance is taken out in the preparation of standard solution from exsiccator, accurately takes by weighing about 20mg in the 100mL volumetric flask.Be dissolution with solvents and dash with 0.1M HCl, shake up to scale.
3. being prepared in of sample solution accurately takes by weighing the about 20mg of phenylalanine sample in the 100mL volumetric flask.With 0.1MHCl is that dissolution with solvents is also dashed to scale, shakes up.
4. the derivatization reaction of standard solution and sample solution is got standard solution and sample solution 1mL respectively with transfer pipet, places the 10mL volumetric flask, adds 1mL 0.5M NaHCO
3Shake up, add 0.5mL 1%DNFB again, shake up.Solution is placed in 60 ℃ of water-baths heating 60 minutes, cooling.Dash to scale with 0.1M phosphate buffer (pH6.5), shake up, prepare HPLC and analyze usefulness.
5. chromatographic condition
Analytical column Alltima C
18, 5 μ m, 250 * 4.6mm
Mobile phase A CN/36mM TEAP, pH2.75 volume ratio: 48/52 isocratic elution
Detect UV360nm
35 ℃ of temperature
Obtain separating resulting with this understanding as chromatogram 6.
6. calculate
The percentage composition of the phenylalanine in the sample=(A
Sample/ A
Standard) * (W
Standard/ W
Standard) * 100%
A
Sample: the peak area of phenylalanine in the sample chromatogram
A
Standard: the peak area of phenylalanine in the standard colors spectrogram
W
Standard: the weighing of phenylalanine primary standard substance, mg
W
Sample: the weighing of phenylalanine base sample, mg.
Claims (8)
1, a kind of amino acid whose analytical approach, the step that it comprises is to use high performance liquid chromatograph, and AA is through 2,4-dinitrofluorobenzene column front derivation, reverse phase separation derivant amino acid-2,4-dinitrofluorobenzene derivant is characterized in that:
With phosphoric acid triethylamine and acetonitrile is eluent gradient wash-out or isocratic elution, reverse phase separation amino acid-2 on analytical column, and 4-dinitrofluorobenzene derivant calculates amino acid whose content with the external standard method quantitative test again.
2, a kind of amino acid whose analytical approach is characterized in that it comprises the steps:
1) prepares amino acid standard solution and sample solution with HCl solution as solvent respectively;
2) get standard solution and sample solution respectively in in the volumetric flask of 10 times of amounts of its volume, add 0.5MNaHCO
3, standard solution or sample solution and NaHCO
3Volume ratio be 1: 1; Add 1%2 again, the acetonitrile solution of 4-dinitrofluorobenzene, with the volume ratio of standard solution or sample solution be 0.5-1: 1, shake up; Place 60C ° of down heating 60 minutes, be cooled to room temperature, with 0.1M pH be 6.5 phosphate buffer towards to scale, shake up, standby;
3) use comprises the HPLC instrument that pump, mixer, constant temperature oven, UV-detector and workstation are formed; At C
18On the analytical column, with phosphoric acid triethylamine/acetonitrile be moving phase, gradient elution or isocratic elution separate AA-2,4-dinitrofluorobenzene; Chromatographic condition is as follows:
Analytical column C
18, 5 μ m, 250 * 4.6mm
Mobile phase A: acetonitrile B:36mM phosphoric acid triethylamine, pH2-3 or 6-7
Type of elution gradient elution or isocratic elution
Detect UV 300-380nm,
Temperature 20-45C °,
4) calculate with the external standard method quantitative test
The concentration of AA=(A in the sample
Sample/ A
Standard) * C
Standard
A
Sample: the peak area of AA in the sample chromatogram
A
Standard: the peak area of AA in the standard colors spectrogram
C
Standard: the concentration of standard solution AA.
3, a kind of amino acid whose analytical approach is characterized in that it comprises the steps:
1) prepares amino acid standard solution and sample solution with 0.1M HCl as solvent respectively;
2) get 1mL standard solution and sample solution respectively in the brown volumetric flask of 10mL, add 1mL 0.5MNaHCO
3, shake up.Add 0.5-1mL 1%2 again, the acetonitrile solution of 4-dinitrofluorobenzene shakes up; Place 60C ° of down heating 60 minutes, be cooled to room temperature, with 0.1M pH be 6.5 phosphate buffer towards to scale, shake up, standby;
3) use comprises the HPLC instrument that pump, mixer, constant temperature oven, UV-detector and workstation are formed.At C
18On the analytical column, with phosphoric acid triethylamine/acetonitrile be moving phase, gradient elution or isocratic elution separate AA-2,4-dinitrofluorobenzene; Chromatographic condition is as follows:
Analytical column C
18, 5 μ m, 250 * 4.6mm
Mobile phase A: acetonitrile B:36mM phosphoric acid triethylamine, pH2-3 or 6-7
Type of elution gradient elution or isocratic elution
Detect UV 360nm
Temperature 35C °;
4) calculate with the external standard method quantitative test
The concentration of AA=(A in the sample
Sample/ A
Standard) * C
Standard
A
Sample: the peak area of AA in the sample chromatogram
A
Standard: the peak area of AA in the standard colors spectrogram
C
Standard: the concentration of standard solution AA.
4, according to claim 2 or 3 said amino acid whose analytical approachs, it is characterized in that the volume ratio of said 36mM phosphoric acid triethylamine and acetonitrile is: 1: 0.01-100.
5,, it is characterized in that said phosphate buffer is K according to claim 2 or 3 said amino acid whose analytical approachs
2HPO
4With KH
2PO
4, or Na
2HPO
4With NaH
2PO
4
6,, it is characterized in that said sample solution is to contain raw material and the preparation thereof that several amino acids is formed, or major component is xylamidine or phenylalanine bulk drug and preparation thereof according to claim 2 or 3 said amino acid whose analytical approachs.
7,, it is characterized in that the said preparation that contains the raw material of several amino acids composition is ephrosis parenteral solution or hepatopathy parenteral solution according to the said amino acid whose analytical approach of claim 6.
8,, it is characterized in that said major component xylamidine preparation is the xylamidine parenteral solution according to the said amino acid whose analytical approach of claim 6.
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