Summary of the invention
The HPLC analytical method that the purpose of this invention is to provide a kind of 2-of mensuration methoxymethyl-4-aminophenol and impurity thereof.
Technical scheme of the present invention is as follows:
The HPLC analytical method of a kind of 2-methoxymethyl-4-aminophenol and impurity thereof is characterized in that adopting reversed-phased high performace liquid chromatographic, and high-efficient liquid phase chromatogram condition is:
Chromatographic column: C
18, 5 μ m, 150 * 4.6mm (I.D.),
The volume ratio that moving phase is formed: acetonitrile: phosphoric acid-phosphate-buffered aqueous solution (PH7.0)=45: 55,
Flow velocity: 0.8ml/min,
Column temperature: 30 ℃,
Detect wavelength: 232nm,
Sample size: 5-10 μ l.
According to HPLC analytical method of the present invention, will measure 50 times of the sample introduction sample concentration dilutions of content, measure the content of 2-methoxymethyl-4-aminophenol main body and impurity thereof, add phosphate 20mmol/l (NH in the moving phase of the present invention
4)
2HPO
4, be 7.0 with phosphoric acid regulating ph value.
Adopt the HPLC analytical method of 2-methoxymethyl-4-aminophenol of the present invention and impurity thereof, when accurately measuring 2-methoxymethyl-4-aminophenol main content, can accurately measure the content of impurity 2-methoxyl methyl-4-nitrophenol, for the Quality Control Analysis of 2-methoxymethyl-4-aminophenol provides the Simple and Reliable analytical approach.
Embodiment
Main experimental apparatus and chromatographic condition:
Waters 1525 type high performance liquid chromatographs, Waters 717plus automatic sampler is joined Waters 2996 type diode array (PDA) detecting devices, and the control of chromatographic fractionation system and record are finished by Waters Empower chromatogram management system.
That chromatographic column is selected for use is C
18, 5 μ m, 150 * 4.6mm (I.D), moving phase: acetonitrile: phosphoric acid-phosphate-buffered aqueous solution (PH=7.0)=45: 55, flow velocity: 0.8ml/min, column temperature: 30 ℃, detect wavelength: 232nm.
Reagent and medicine:
2-methoxymethyl-4-aminophenol reference substance (>99.0%)
2-methoxyl methyl-4-nitrophenol reference substance (>99.0%)
Acetonitrile (HPLC level);
Phosphate-buffered aqueous solution (PH=7.0)
Moving phase and solution with water are the quartzy distilled water of secondary in the experiment.
Embodiment 1: the selection of high-efficient liquid phase chromatogram condition
1.1 the selection of moving phase
Acetonitrile is used in experiment: the moving phase isocratic elution of water=45: 55,2-methoxymethyl-4-aminophenol and impurity separation case are good.But because amino in the 2-methoxymethyl-4-aminophenol and hydroxyl can interact with the silicon hydroxyl of remnants in the chromatographic column, caused 2-methoxymethyl-4-aminophenol chromatographic peak that phenomenon (Fig. 1-a), influence the accuracy of quantitative test 2-methoxymethyl-4-aminophenol of hangover is arranged.Phosphate 20mmol/l (NH is added in employing in moving phase
4)
2HPO
4And be 7.0 method with phosphoric acid regulating ph value, can eliminate conditions of streaking (Fig. 1-b) of chromatographic peak.
1.2 detect the selection of wavelength
Carry out continuous sweep with diode array detector at 200-400nm, the 2-methoxymethyl-4-aminophenol has characteristic absorption (Fig. 2-a), and 2-methoxyl methyl-4-nitrophenol has characteristic absorption (Fig. 2-b) at 229nm and 318nm place at 235nm and 303nm place.Take into account each component sensitivity, selecting to detect wavelength is 232nm.
1.3 the selection of sample concentration
In order to take into account the mensuration of major component content and impurity content, when measuring impurity, the concentration of sample must be enough big, so that impurity can go out the peak; When measuring major component, solution concentration is crossed conference and is caused main peak to exceed the range of linearity and make quantitatively inaccurately, so measure behind 50 times of the diluted samples again, tests accuracy and sees Table 1.
Embodiment 2: the preparation of standard solution and sample solution
Accurate weighing 50.00mg 2-methoxymethyl-4-aminophenol reference substance, place the 50ml volumetric flask, with moving phase dissolving and be diluted to scale, shake up, being made into concentration is the standard reserving solution of the 2-methoxymethyl-4-aminophenol of 1.0mg/ml, and stepwise dilution is to desired concn during use.
Accurately take by weighing 25.00mg 2-methoxyl methyl-4-nitrophenol reference substance, place the 50ml volumetric flask, with moving phase dissolving and be diluted to scale, shake up, getting concentration is the standard reserving solution of 2-methoxyl methyl-4-nitrophenol of 0.5mg/ml, and stepwise dilution is to desired concn during use.
Embodiment 3: the making of typical curve
High-efficient liquid phase chromatogram condition: chromatographic column is C
18, 5 μ m, 150 * 4.6mm (I.D); The volume ratio that moving phase is formed: acetonitrile: phosphoric acid-phosphate-buffered aqueous solution (PH7.0)=45: 55; Flow velocity 0.8ml/min; 30 ℃ of column temperatures; Detect wavelength 232nm.Get 2-methoxymethyl-4-aminophenol standard solution 10 μ l sample introductions, with peak area to concentration mapping (Fig. 3).The 2-methoxymethyl-4-aminophenol is linear in the scope of solution concentration 0.0001~1.0mg/ml, and regression equation is:
A
The 2-methoxymethyl-4-aminophenol=193754.33232+3.31341E7C, r=0.99997
Get 2-methoxyl methyl-4-nitrophenol standard solution 10 μ l sample introductions, with peak area to concentration mapping (Fig. 4).2-methoxyl methyl-4-nitrophenol is linear in solution concentration is the scope of 0.0001~0.10mg/ml, and regression equation is:
A
2-methoxyl methyl-4-nitrophenol=12476.47946+1.79587E7C, r=0.99953
When signal to noise ratio (S/N ratio) S/N=3, the lowest detectable limit that records 2-methoxymethyl-4-aminophenol and 2-methoxyl methyl-4-nitrophenol is respectively 0.00001mg/ml and 0.00002mg/ml.
Embodiment 4: the mensuration of sample
4.1 main Determination on content
Accurately take by weighing 50.0mg 2-methoxymethyl-4-aminophenol sample, place the 50ml volumetric flask, with moving phase dissolving and be diluted to scale, shake up, concentration be the solution of 1.0mg/ml.Get above-mentioned 1.0mg/ml solution 1.0ml and place the 50ml volumetric flask, add moving phase and be diluted to scale, shake up, get the solution of 0.02mg/ml, standby.
Select the sample of any 5 lot numbers, prepare the 0.02mg/ml sample solution as stated above.High-efficient liquid phase chromatogram condition, chromatographic column C
18, 5 μ m, 150 * 4.6mm (I.D); The volume ratio that moving phase is formed: acetonitrile: phosphoric acid-phosphate-buffered aqueous solution (PH7.0)=45: 55; Flow velocity 0.8ml/min; 30 ℃ of column temperatures; Detect wavelength 232nm and carry out HPLC and analyze, sample size is 10 μ l (Fig. 1-b).
4.2 the mensuration of impurity content
Select the sample of any five lot numbers, prepare the 1.0mg/ml sample solution as stated above.High-efficient liquid phase chromatogram condition: chromatographic column C
18, 5 μ m, 150 * 4.6mm (I.D); The volume ratio that moving phase is formed: acetonitrile: phosphoric acid-phosphate-buffered aqueous solution (PH7.0)=45: 55; Flow velocity is 0.8ml/min; Column temperature is 30 ℃; Detecting wavelength is that 232nm carries out the HPLC analysis, and sample size is 10 μ l (Fig. 5).
The testing result of major component 2-methoxymethyl-4-aminophenol and impurity 2-methoxyl methyl-4-nitrophenol is listed in table 1 in the sample.
Table 1. testing result
Lot number |
The 2-methoxymethyl-4-aminophenol |
2-methoxyl methyl-4-nitrophenol |
Content % |
RSD% |
Content % |
RSD% |
1 |
99.30 |
0.45 |
0.29 |
1.5 |
2 |
99.26 |
0.48 |
0.33 |
1.8 |
3 |
99.23 |
0.37 |
0.38 |
2.0 |
4 |
99.28 |
0.53 |
0.34 |
2.1 |
5 |
99.21 |
0.34 |
0.27 |
1.5 |