CN103122023A - Purification method for triptorelin - Google Patents
Purification method for triptorelin Download PDFInfo
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- CN103122023A CN103122023A CN2013100741540A CN201310074154A CN103122023A CN 103122023 A CN103122023 A CN 103122023A CN 2013100741540 A CN2013100741540 A CN 2013100741540A CN 201310074154 A CN201310074154 A CN 201310074154A CN 103122023 A CN103122023 A CN 103122023A
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Abstract
The invention provides a purification method for triptorelin, which comprises the following steps: firstly, dissolving a sample by using an aqueous solution containing organic solvents, and carrying out gradient elution on the sample by taking octadecylsilane chemically bonded silica as a stationary phase, taking buffer salts of a phosphoric acid or sulfuric acid as a phase A, and taking acetonitrile as a phase B; then, converting triptorelin phosphates into acetates by using a reversed-phase HPLC (high performance liquid chromatography) method and taking octadecylsilane chemically bonded silica as a stationary phase, and freeze-drying the obtained acetates, so that triptorelin acetates with a purity of more than 99.7% and an individual impurity of less than 99.7% of are obtained, and the total yield is as high as over 30%. The invention provides a triptorelin purification process which is low in cost, high in yield, simple in technological process, less in sample impurities, stable and controllable, and beneficial to the implementation of industrialization.
Description
Technical field
The present invention relates to a kind of purification process of polypeptide drugs, the particularly purification process of triptorelin belongs to the pharmaceutical chemistry field.
Background technology
Triptorelin, English name: Triptorelin, commodity look ammonia by name Rayleigh, Wy-42462, Decapeptyl etc., it is a kind of gonadotropin releasing hormone (GnRH) decapeptide congener that 10 amino acid form, its similar goserelin, its peptide order is: Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH
2, its embonate of clinical use (storage preparation).
Triptorelin is as a kind of Amino-Cerv, and its indication is mainly: 1. the palliative treatment of advanced prostate cancer; 2. sexual prematurity; 3. endometriosis, female infertility and hysteromyoma.When continuing medication with therapeutic dose, this product can suppress the secretion of short sexual hormoue forcefully, and the natural GnRH of its specific activity is strong.After the beginning administration, the LH during blood follows, FSH, the transience peak can appear in the level of testosterone and estradiol.After continuous long term administration, generally in the synthetic obviously minimizing of the steroid of the secretory volume continuous decrease that can be observed LH and FSH 2～4 weeks of begin treatment and testis and ovary.Triptorelin is to treat at present the optimal medicine of central sexual prematurity, can suppress quickly and effectively the maturation of secondal sexual character and the speed that health is linearly grown, and after drug withdrawal, the natural process of Development in Puberty biology is unaffected.Clinical sexual prematurity, endometriosis, female acyesis and the hysteromyoma etc. of being mainly used in.
Prior art CN101357936A discloses a kind of separation purification method of triptorelin, concrete steps are as follows: the triptorelin crude product is dissolved in 5% acetic acid, filters, filtrate is through the C18 column purification, moving phase: 0.1MNH4Ac: acetonitrile (7.5: 2.5, volume ratio); Flow velocity is 500ml/min; The detection wavelength is: 280nm; Follow the tracks of with liquid chromatograph and collect needed effluent liquid, desalt after sample peak merging, freeze-drying approximately gets 20g white block finished product (MW:1311.5,15.25mmol), total recovery 25.4% (in the 60mmol of resin).This separation purification method is difficult to control the impurity of raw material limits the quantity of, and is difficult to reach medicinal required purity.
Limit the quantity of for the impurity of effectively controlling the triptorelin raw material, improve purity, need the further new separation purification method of research.
Summary of the invention
The present invention is directed to the physicochemical characteristics of triptorelin, provide a kind of with low cost, high yield, technological process is simple, sample impurity is few and it is controlled to stablize, be conducive to realize the triptorelin purifying process of industrialization.
For achieving the above object, the present invention takes following technical scheme: a kind of purification process of triptorelin is characterized in that:
1) with the aqueous solution of the organic solvent of volume ratio content 5%-15%, according to concentration 100g/L dissolving triptorelin sample, the chromatographic column take octadecylsilane chemically bonded silica as stationary phase is take buffering salt as the A phase, acetonitrile is the B phase, carries out gradient elution (this step aftermentioned is referred to as " purifying ");
2) adopt Reversed phase HPLC method, take octadecylsilane chemically bonded silica as stationary phase, with triptorelin phosphoric acid salt change into acetate, freeze-drying obtains triptorelin (this step aftermentioned is referred to as " turning salt ").
Step 1), in described step 1) preferably with the aqueous solution of the organic solvent of volume ratio content 8%-10%, according to concentration 100g/L dissolving triptorelin sample, wherein, described dissolution sample organic solvent used is the stronger organic reagent of polarity, comprise methyl alcohol, acetonitrile, dimethyl sulfoxide (DMSO) (DMSO), Virahol etc., wherein preferred acetonitrile.
The applicant finds, due to the feature that this body structure of triptorelin forms, easily contains various not exclusively synthetic or racemization impurity in crude product, and the solubilising reagent of therefore thick peptide sample requires very harsh.Find after applicant's repetition test, organic solvent content is in 5%～15% scope the time, and sample dissolution is better, and wherein organic solvent content is when 8%～10% scope, and effect is more obvious; And find when groping different organic reagent, the organic reagent of the stronger polarity such as DMSO, methyl alcohol, acetonitrile, Virahol is better to the solute effect of sample, wherein again with the acetonitrile best results.
Wherein, described 1), the buffering salt of A phase is phosphate-buffered salt or sulfuric acid buffering salt.
Wherein, described phosphate-buffered salt is 0.1%-0.8%(v/v) phosphate buffered saline buffer, and the solvent of damping fluid is water; Described sulfuric acid buffer salt system is 0.2%-0.6%(v/v) vitriol damping fluid.
Wherein, described phosphate-buffered salt comprises the buffer salt system such as phosphoric acid triethylamine, Sodium phosphate dibasic, dipotassium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate; The sulfuric acid buffer salt system is the ammonium sulfate buffering salt.
In triptorelin was carried out purge process, the applicant analyzed, contrasts the mobile phase system of difference, looks for best moving phase system.Find in process, most of moving phase systems can only be simple removes partial impurities, once in purge process in order to guarantee purity, can reach just often will repeatedly carry out repeatedly loaded down with trivial details recovery purifying the purity that meets the requirements; Through repeatedly groping of applicant, final 0.1%-0.8%(v/v) phosphate buffer and 0.2%-0.6%(v/v) vitriol buffer system, to removing the impurity successful in thick peptide, only purifying of need, once recovery, save the multistep repetitive operation.Purifying can be obtained purity greater than 95%, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.
Wherein, described phosphate-buffered salt and sulfuric acid buffering salt are transferred pH1.5-3.2 with sodium hydroxide or potassium hydroxide or ammoniacal liquor.The applicant finds, triptorelin is for the requirement of pH value harshness comparatively, and the pH value was higher than 5 o'clock, and sample is easily separated out, and the pH value is lower than 1 o'clock, the sample stability variation.Through repetition test, when finding pH2.0-3.0, sample stability is best.
In step 1), described wash-out is B phase 20%-40%(v/v with the gradient of reversed-phase HPLC), linear gradient elution time 60min.
Step 2), wherein, the described salt that turns comprises two steps: turn salt with the acetate buffer salts solution; Rear is the A phase with aqueous acetic acid, and acetonitrile is that B carries out gradient elution mutually.
Wherein, the acetate buffer salts solution is the aqueous solution of acetate buffer salt and the mixing solutions of acetonitrile, and in the aqueous solution of described acetate buffer salt, acetate buffer salt is ammonium acetate; The aqueous solution of acetate buffer salt and the volume ratio of acetonitrile are 95:5.
Wherein, the concentration of described acetate buffer salt is 0.02-0.1%(v/v), the pH value scope of acetate buffer salt is 1.5-4.5.The applicant finds, the acetate buffer salt concn is lower than 0.02% the time, and higher than 0.1% the time, sample is all unstable.When acetate buffer salt pH value scope was 1.5-4.5, sample was all very stable.
Wherein, in aqueous acetic acid, the volume by volume concentration of acetic acid is 0.02%-0.65%, and the gradient of described reversed-phase HPLC is B%:5%～25%, and the linear gradient elution time is 30min.The applicant finds, because the first step sample purity is high, therefore 30min in turning the salt process, but just purifying obtain purity up to 99.8%, maximumly singly assortedly obviously shortened less than 0.02% triptorelin acetate the time that turns the salt purifying, saved production cost.
What described reversed-phase column was used is that octadecylsilane chemically bonded silica is the chromatographic column of stationary phase.Also be referred to as volume ratio in specification sheets in (v/v), the ratio of two kinds of solvents of equal volume unit is as 0.02%(v/v) aqueous acetic acid refers to that volume is the mixing solutions of 0.02L acetic acid and 1L water.
Present method is by two step purifying, remove respectively distance in the triptorelin crude product assorted make purity greater than 99.7%, single assorted less than 0.03% and change into stable acetate, process stabilizing is controlled, purity is high, productive rate is high, has practical value and application prospect widely.
Embodiment
Embodiment 1
Embodiment 1 the first step purifying
Acetonitrile solution with volume ratio 5% dissolves thick peptide according to the concentration of 100g/L, stirs to make sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: 0.1% phosphate buffered (v/v), transfer pH2.0 with sodium hydroxide; B phase: acetonitrile, flow velocity: 60-80ml/min, gradient: B%:20%～40%, linear gradient elution 60min; Detect wavelength: 230nm.Sample size is 1.5g.Collection purpose peak can obtain purity greater than 95% with purifying, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.Satisfactory triptorelin solution is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 2 the first step purifying
The DMSO aqueous solution with volume ratio 15% dissolves thick peptide according to the concentration of 100g/L, stirs to make sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: 0.8% phosphate buffered (v/v), transfer pH3.0 with ammoniacal liquor; B phase: acetonitrile, flow velocity: 60-80ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Detect wavelength: 230nm.Sample size is 1.5g.Collection purpose peak can obtain purity greater than 95% with purifying, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.The satisfactory bent triptorelin solution of collecting is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 3 the first step purifying
Isopropanol water solution with volume ratio 10% dissolves thick peptide according to the concentration of 100g/L, stirs to make sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: 0.2% vitriol buffering (v/v), transfer pH2.0 with phosphoric acid; B phase: acetonitrile, flow velocity: 60-80ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Detect wavelength: 230nm.Sample size is 1.5g.Collection purpose peak can obtain purity greater than 95% with purifying, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.The satisfactory bent triptorelin solution of collecting is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 4 the first step purifying
Acetonitrile solution with volume ratio 15% dissolves thick peptide according to the concentration of 100g/L, stirs to make sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm * 25cm.Moving phase: A phase: 0.6% vitriol buffering (v/v), transfer pH3.0 with phosphoric acid; B phase: acetonitrile, flow velocity: 200-250ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Detect wavelength: 230nm.Sample size is 15-20g.Collection purpose peak can obtain purity greater than 95% with purifying, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.The satisfactory bent triptorelin solution of collecting is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 5 the first step purifying
With the concentration dissolving thick peptide of volume ratio 8% methanol aqueous solution according to 100g/L, stirring makes sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm * 25cm.Moving phase: A phase: 0.5% phosphate buffered (v/v), transfer pH2.5 with sodium hydroxide; B phase: acetonitrile, flow velocity: 220-250ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Detect wavelength: 230nm.Sample size is 15-20g.Collection purpose peak can obtain purity greater than 95% with purifying, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.The satisfactory bent triptorelin solution of collecting is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 6 the first step purifying
Isopropanol water solution with volume ratio 5% dissolves thick peptide according to the concentration of 100g/L, stirs to make sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm * 25cm.Moving phase: A phase: 0.5% vitriol buffering (v/v), transfer pH2.3 with phosphoric acid; B phase: acetonitrile, flow velocity: 220-250ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Detect wavelength: 230nm.Sample size is 15-20g.Receive purifying and can obtain purity greater than 95%, list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.Satisfactory bent Ji Mudefeng, the triptorelin solution collected is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 7 the first step purifying
The DMSO aqueous solution with volume ratio 8% dissolves thick peptide according to the concentration of 100g/L, stirs to make sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm * 30cm.Moving phase: A phase: 0.3% phosphate buffered (v/v), transfer pH2.5 with potassium hydroxide; B phase: acetonitrile, flow velocity: 450-550ml/min, gradient: B%:20%-40%, linear gradient elution 30min; Detect wavelength: 230nm.Sample size is 20-25g.Collection purpose peak can obtain purity greater than 95% with purifying, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.The satisfactory bent triptorelin solution of collecting is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 8 the first step purifying
Methanol aqueous solution with volume ratio 6% dissolves thick peptide according to the concentration of 100g/L, stirs to make sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm * 30cm.Moving phase: A phase: 0.8% phosphate buffered (v/v), transfer pH3.0 with ammoniacal liquor; B phase: acetonitrile, flow velocity: 450-550ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Detect wavelength: 230nm.Sample size is 20-25g.Collection purpose peak can obtain purity greater than 95% with purifying, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.The satisfactory bent triptorelin solution of collecting is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 9 the first step purifying
Acetonitrile solution with volume ratio 5% dissolves thick peptide according to the concentration of 100g/L, stirs to make sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm * 30cm.Moving phase: A phase: 0.5% phosphate buffered (v/v), transfer pH2.0 with potassium hydroxide; B phase: acetonitrile, flow velocity: 1900-2200ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Detect wavelength: 230nm.Sample size is 60-85g.Collection purpose peak can obtain purity greater than 95% with purifying, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.The satisfactory bent triptorelin solution of collecting is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 10 the first step purifying
The DMSO aqueous solution with volume ratio 15% dissolves thick peptide according to the concentration of 100g/L, stirs to make sample dissolve the rear membrane filtration of using fully, collects filtrate.
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm * 30cm.Moving phase: A phase: 0.2% vitriol buffering (v/v), transfer pH2.5 with phosphoric acid; B phase: acetonitrile, flow velocity: 1900-2200ml/min, gradient: B%:20-40%, linear gradient elution 60min; Detect wavelength: 230nm.Sample size is 60-85g.Collection purpose peak can obtain purity greater than 95% with purifying, and list turns salt after mixing and removing most of acetonitrile less than 0.3% sample.Purity less than 95% greater than 80% once partially recycled, get its moderate purity greater than 95%, single assorted less than 0.3% part and other salable product one salt that runs up, reclaim purity undesirable discarded.The satisfactory bent triptorelin solution of collecting is standby after vacuum rotary steam under the condition of 30 ℃ of water temperatures is concentrated into approximately 15mg/mL.
Embodiment 11 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: 0.02% aqueous acetic acid (v/v); B phase: acetonitrile; The C phase: 0.02% Ammoniom-Acetate, pH2.0, flow velocity: 80ml/min detects wavelength: 230nm.Sample size is 1.5g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steam under 30 ℃ of conditions of water temperature of implementing 1 collection approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.75% triptorelin, other impurity is all less than 0.02%.
Embodiment 12 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: 0.05% aqueous acetic acid (v/v); The B phase: acetonitrile, the C phase: 0.05% Ammoniom-Acetate, pH2.5, flow velocity: 60ml/min detects wavelength: 230nm.Sample size is 1.5g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steams under 30 ℃ of conditions of water temperature of implementing 2 collections approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.78% triptorelin, other impurity is all less than 0.02%.
Embodiment 13 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: 0.1% aqueous acetic acid (v/v); B phase: acetonitrile, C phase: 0.08% Ammoniom-Acetate, pH3.0, flow velocity: 80ml/min; Detect wavelength: 230nm.Sample size is 1.5g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steams under 30 ℃ of conditions of water temperature of implementing 3 collections approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.76% triptorelin, other impurity is all less than 0.02%.
Embodiment 14 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm * 25cm.Moving phase: A phase: 0.06% aqueous acetic acid (v/v); The B phase: acetonitrile, flow velocity: 220ml/min detects wavelength: 230nm.Sample size is 10-15g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steams under 30 ℃ of conditions of water temperature of implementing 4 collections approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.76% triptorelin, other impurity is all less than 0.02%.
Embodiment 15 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm * 25cm.Moving phase: A phase: 0.03% aqueous acetic acid (v/v); The B phase: acetonitrile, the C phase: 0.10% Ammoniom-Acetate, pH4.5, flow velocity: 250ml/min detects wavelength: 230nm.Sample size is 15-20g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steams under 30 ℃ of conditions of water temperature of implementing 5 collections approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.72% triptorelin, other impurity is all less than 0.02%.
Embodiment 16 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm * 30cm.Moving phase: A phase: 0.09% aqueous acetic acid (v/v); The B phase: acetonitrile, the C phase: 0.07% Ammoniom-Acetate, pH3.2, flow velocity: 400ml/min detects wavelength: 230nm.Sample size is 15-20g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steams under 30 ℃ of conditions of water temperature of implementing 6 collections approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.75% triptorelin, other impurity is all less than 0.02%.
Embodiment 17 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm * 30cm.Moving phase: A phase: 0.65% aqueous acetic acid (v/v); The B phase: acetonitrile, the C phase: 0.06% Ammoniom-Acetate, pH2.0, flow velocity: 450ml/min detects wavelength: 230nm.Sample size is 15-20g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steams under 30 ℃ of conditions of water temperature of implementing 7 collections approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.78% triptorelin, other impurity is all less than 0.02%.
Embodiment 18 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm * 30cm.Moving phase: A phase: 0.32% aqueous acetic acid (v/v); The B phase: acetonitrile, the C phase: 0.05% Ammoniom-Acetate, pH2.5, flow velocity: 1900ml/min detects wavelength: 230nm.Sample size is 60-85g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steams under 30 ℃ of conditions of water temperature of implementing 8 collections approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.75% triptorelin, other impurity is all less than 0.02%.
Embodiment 19 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm * 30cm.Moving phase: A phase: 0.10% aqueous acetic acid (v/v); The B phase: acetonitrile, the C phase: 0.02% Ammoniom-Acetate, pH2.0, flow velocity: 2200ml/min detects wavelength: 230nm.Sample size is 60-85g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steams under 30 ℃ of conditions of water temperature of implementing 9 collections approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.74% triptorelin, other impurity is all less than 0.02%.
Embodiment 20 turns salt
Chromatographic condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm * 30cm.Moving phase: A phase: 0.08% aqueous acetic acid (v/v); The B phase: acetonitrile, the C phase: 0.68% Ammoniom-Acetate, pH2.3, flow velocity: 2000ml/min detects wavelength: 230nm.Sample size is 60-85g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A～25%B+75%A, linear gradient elution 30min; Collection purpose peak is concentrated into triptorelin solution vacuum rotary steams under 30 ℃ of conditions of water temperature of implementing 10 collections approximately and goes to the 50ml cillin bottle after 50-200mg/mL.Can obtain purity after lyophilize greater than 99.75% triptorelin, other impurity is all less than 0.02%.
Claims (9)
1. the purification process of a triptorelin:
Step 1): with the aqueous solution of the organic solvent of volume ratio content 5%-15%, according to concentration 100g/L dissolving triptorelin sample, chromatographic column take octadecylsilane chemically bonded silica as stationary phase, be the A phase with the phosphate-buffered salt of volume ratio 0.1%-0.8% or the sulfuric acid buffer salt solution of volume ratio 0.2%-0.6% with adjusting PH with base 1.5-3.2, acetonitrile is that B carries out the gradient elution purifying mutually, and gradient is B%:20%-40%;
Step 2): turn salt with the acetate buffer salts solution; Chromatographic column take octadecylsilane chemically bonded silica as stationary phase is carried out the gradient elution purifying, and rear is the A phase with aqueous acetic acid, and acetonitrile is that B carries out gradient elution mutually.
2. method according to claim 1, it is characterized in that: in described step 1) with the aqueous solution of the organic solvent of volume ratio content 8%-10%, according to concentration 100g/L dissolving triptorelin sample, described organic solvent is methyl alcohol, acetonitrile, dimethyl sulfoxide (DMSO) or Virahol.
3. method according to claim 1, it is characterized in that: in described step 1), phosphate-buffered salt is triethylenetetraminehexaacetic acid amine, Sodium phosphate dibasic, dipotassium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC or potassium primary phosphate buffer salt system; The sulfuric acid buffering salt is the ammonium sulfate buffering salt; Described alkali is sodium hydroxide, potassium hydroxide or ammoniacal liquor.
4. method according to claim 2, it is characterized in that: in described step 1), phosphate-buffered salt is triethylenetetraminehexaacetic acid amine, Sodium phosphate dibasic, dipotassium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC or potassium primary phosphate buffer salt system; The sulfuric acid buffering salt is the ammonium sulfate buffering salt; Described alkali is sodium hydroxide, potassium hydroxide or ammoniacal liquor.
5. the described method of arbitrary claim of according to claim 1 to 4, it is characterized in that: described step 2), the acetate buffer salts solution is the aqueous solution of acetate buffer salt and the mixing solutions of acetonitrile, in the aqueous solution of described acetate buffer salt, acetate buffer salt is ammonium acetate, and the volume by volume concentration of acetate buffer salt is 0.02-0.1%; The aqueous solution of acetate buffer salt and the volume ratio of acetonitrile are 95:5.
6. method according to claim 5, it is characterized in that: described step 2), the acetate buffer salts solution is the aqueous solution of acetate buffer salt and the mixing solutions of acetonitrile, and the pH value scope of the aqueous solution of described acetate buffer salt is 1.5-4.5.
7. the described method of arbitrary claim of according to claim 1 to 4, it is characterized in that: described step 2) in aqueous acetic acid, the volume by volume concentration of acetic acid is 0.02%-0.65%, gradient is B%:5%～25%, linear gradient elution 30min.
8. method according to claim 5, it is characterized in that: described step 2) in aqueous acetic acid, the volume by volume concentration of acetic acid is 0.02%-0.65%, gradient is B%:5%-25%, linear gradient elution 30min.
9. method according to claim 6, it is characterized in that: described step 2) in aqueous acetic acid, the volume by volume concentration of acetic acid is 0.02%-0.65%, gradient is B%:5%-25%, linear gradient elution 30min.
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