JP2000351791A - Purification of doxorubicin - Google Patents
Purification of doxorubicinInfo
- Publication number
- JP2000351791A JP2000351791A JP11161030A JP16103099A JP2000351791A JP 2000351791 A JP2000351791 A JP 2000351791A JP 11161030 A JP11161030 A JP 11161030A JP 16103099 A JP16103099 A JP 16103099A JP 2000351791 A JP2000351791 A JP 2000351791A
- Authority
- JP
- Japan
- Prior art keywords
- doxorubicin
- aqueous solution
- solution
- hydrochloric acid
- purity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、アントラサイクリ
ン抗生物質ドキソルビシンの高純度製品を得るための精
製方法に関する。The present invention relates to a method for purifying an anthracycline antibiotic doxorubicin to obtain a highly purified product.
【０００２】[0002]
【従来の技術】ドキソルビシン（アドリアマイシンとも
称されている）は、放線菌の培養液あるいはダウノマイ
シンを原料とした化学合成により得られることが知られ
ており、実験腫瘍に対して広域抗癌スペクトルを有する
アントラサイクリン抗生物質である（米国特許第３,５
９０,０２８号明細書参照）。そして、現に、癌化学療
法剤として臨床的にも広く利用されている。2. Description of the Related Art Doxorubicin (also called adriamycin) is known to be obtained by actinomycete culture or chemical synthesis using daunomycin as a raw material, and has a broad anticancer spectrum against experimental tumors. Is an anthracycline antibiotic (U.S. Pat.
90,028). And it is actually widely used clinically as a cancer chemotherapeutic agent.
【０００３】ドキソルビシン（以下、「ＤＸ」と略記す
る）を初めとする類似のアントラサイクリン抗生物質
は、前記培養液あるいは反応液から粗精製したものを、
カチオン交換樹脂［例、Amberlite（商標）ＩＲＣ５
０］にかけ、塩化ナトリウム含有水または水と塩化ナト
リウムを含有するメタノールで溶離することにより、さ
らに精製されている（特公昭４９−４４３４７号、特開
昭５０−１５８８０号）。また、重合体状イオン交換樹
脂［例、Amberlite（商標）ＥＲ１８０］もしくはＣＭ
セファロース樹脂［例、Sepharose（商標）ＣＨＢ］に
前記抗生物質を吸着させた後、酸性の水と極性溶媒の混
合物を用いて溶離する工程を含む方法も公表されている
［特開昭５９−１１８７９７号ならびにそれに由来する
特公平４−３９４７６号：これらに対応する米国特許第
４,８６１,８７０号］。さらに、後者の公報には、上記
重合体状イオン交換樹脂で処理する前の予備精製工程に
おいて吸着性多孔質合成樹脂担体［例、Amberlite（商
標）ＸＡＤ２］に粗精製ＤＸを吸着させた後、水：メタ
ノール（７：３）（ｖ／ｖ）混合物を用いて前記抗生物
質を溶離することも記載されているが、溶離液はｐＨ調
整していない。しかも高純度のＤＸを得るためにはさら
なる精製を必要としている。[0003] Similar anthracycline antibiotics such as doxorubicin (hereinafter abbreviated as "DX") are obtained by roughly purifying the aforementioned culture solution or reaction solution.
Cation exchange resin [eg, Amberlite ™ IRC5
0] and eluted with sodium chloride-containing water or water and methanol containing sodium chloride (JP-B-49-44347, JP-A-50-15880). Also, a polymer ion exchange resin [eg, Amberlite (trademark) ER180] or CM
A method is also disclosed which comprises a step of adsorbing the above-mentioned antibiotic on a Sepharose resin [eg, Sepharose (trademark) CHB] and then eluting with a mixture of acidic water and a polar solvent [JP-A-59-118797]. And Japanese Patent Publication No. 4-39476 derived therefrom: U.S. Pat. No. 4,861,870 corresponding thereto]. Further, the latter publication discloses that after a crude purified DX is adsorbed on an adsorptive porous synthetic resin carrier [eg, Amberlite (trademark) XAD2] in a pre-purification step before treatment with the polymeric ion exchange resin, Elution of the antibiotic with a water: methanol (7: 3) (v / v) mixture is also described, but the eluent is not pH adjusted. Moreover, further purification is required to obtain high-purity DX.
【０００４】以上の従来技術の吸着、溶離手段によれ
ば、一定の高純度ＤＸを得ることもできるようである
が、高純度製品を得ようとすればする程、目的とするＤ
Ｘの回収率が低下し、逆に回収率を高めようとすればす
る程、製品の純度を高めることができない。特に、培養
液あるいは培養液の粗精製液からＤＸを回収しようとす
る場合、その後の結晶化を初めとする精製工程により除
去することが困難な不純物（たとえば、後述するＨＰＬ
Ｃ分析でＤＸより保持時間の大きいもの）が精製ＤＸに
随伴してくる傾向がある。[0004] According to the above-mentioned conventional adsorption and elution means, it is possible to obtain a certain high purity DX.
As the recovery rate of X decreases and the recovery rate increases, the purity of the product cannot be increased. Particularly, when DX is to be recovered from a culture solution or a crudely purified solution of a culture solution, impurities which are difficult to remove by a purification step such as subsequent crystallization (for example, HPL described later)
(Having a longer retention time than DX in C analysis) tends to accompany the purified DX.
【０００５】[0005]
【発明が解決しようとする課題】したがって本発明の目
的は、出発粗精製ＤＸから高純度ＤＸを高い回収率で、
しかも簡便な方法で得ることのできる精製方法を提供す
ることにある。Accordingly, it is an object of the present invention to provide high-purity DX from a starting crudely-purified DX with a high recovery rate.
It is another object of the present invention to provide a purification method which can be obtained by a simple method.
【０００６】[0006]
【課題を解決するための手段】本発明者らは、高純度製
品を得るためのイオン交換樹脂［例、Amberlite（商
標）ＥＲ１８０］を用いる処理に先立って、予備精製の
工程で使用することが従来提案されていた吸着性（もし
くは疎水性）合成樹脂担体を用い、特定の溶離（または
溶出）条件を選ぶことにより、高回収率でかつ高純度の
ＤＸが得られることを見い出した。たとえば、上記特開
昭５９−１１８７９７号公報に記載されているように、
高純度製品を得るためには、予備精製工程である疎水性
合成樹脂工程とイオン交換樹脂工程の併用が必須である
が、予備精製工程で使用されていたにすぎない疎水性合
成樹脂担体の使用だけで、上記課題が解決できることは
全く驚くべきことであろう。SUMMARY OF THE INVENTION The present inventors have found that prior to treatment with an ion exchange resin (eg, Amberlite ™ ER180) to obtain a high purity product, it may be used in a pre-purification step. It has been found that by using a conventionally proposed adsorptive (or hydrophobic) synthetic resin carrier and selecting specific elution (or elution) conditions, DX with high recovery and high purity can be obtained. For example, as described in JP-A-59-118797,
In order to obtain a high-purity product, it is essential to use a hydrophobic synthetic resin step, which is a pre-purification step, and an ion-exchange resin step, but use of a hydrophobic synthetic resin carrier, which was only used in the pre-purification step It would be quite surprising that the above problem could be solved by itself.
【０００７】したがって本発明によれば、粗精製ＤＸか
らＤＸを吸着させた疎水性多孔質合成樹脂担体を、水混
和性有機溶媒を含有するｐＨ２.５〜５に調整された水
性溶液による溶出操作にかけることによりＤＸを溶出
し、こうして得られた溶出液から精製されたＤＸを回収
することを特徴とするＤＸの精製方法が提供される。Therefore, according to the present invention, a process for eluting a hydrophobic porous synthetic resin carrier adsorbing DX from crudely purified DX with an aqueous solution containing a water-miscible organic solvent and adjusted to pH 2.5-5. The method of the present invention provides a method for purifying DX, wherein DX is eluted by subjecting to DX and the purified DX is recovered from the eluate thus obtained.
【０００８】本発明にいう粗精製ＤＸ（ＤＸ）は、それ
を含有する培養液から、それ自体既知の分離もしくは精
製方法に従って菌体その他の不純物が除去され、ＤＸが
ある程度濃縮されているものであれば、ＤＸの含有率は
いかなるものであってもよい。このような分離もしくは
精製方法には上記従来技術として紹介した方法をも包含
される。また、発酵生産物であるダウノマイシンを原料
とし、その１４位をハロゲン化し、さらにこれを加水分
解する半合成工程を経て得られる一定の不純物を随伴す
るＤＸ含有処理物をも、本発明にいう粗精製ＤＸに包含
される。[0008] The partially purified DX (DX) according to the present invention is obtained by removing bacterial cells and other impurities from a culture solution containing the DX according to a separation or purification method known per se, and DX is concentrated to some extent. If so, the DX content may be any. Such separation or purification methods include the methods introduced as the above-mentioned prior art. In addition, the DX-containing treated product obtained from a fermentation product daunomycin as a raw material, which undergoes a semi-synthesis step of halogenating the 14th position thereof, and further hydrolyzing it, is also referred to in the present invention as a crude product. Included in purified DX.
【０００９】しかしながら、本発明方法がより効率的に
適用できるのは、ＤＸの相対純度が約８０％以上、好ま
しくは９０〜９８％の粗精製ＤＸに向けられる場合であ
る。本明細書で使用する場合の「相対純度」とは、試料
を後述するようなＨＰＬＣ分析した結果得られる各成分
の溶出挙動を示す総ピーク面積に対するＤＸ由来のピー
ク面積の割合を意味する。However, the method of the present invention can be more efficiently applied when the relative purity of DX is about 80% or more, preferably 90 to 98%, for crude DX. As used herein, the term "relative purity" means the ratio of the peak area derived from DX to the total peak area indicating the elution behavior of each component obtained as a result of HPLC analysis of a sample as described below.
【００１０】粗精製ＤＸを吸着させるのに使用する疎水
性多孔質合成樹脂担体としては、本発明の目的に沿う性
能を有するものであればどの種類のものであってもよい
が、具体的にはスチレンとジビニルベンゼンから特殊な
方法で重合して得られる多孔質（もしくは多孔性）ポリ
マーを挙げることができる。このような多孔質ポリマー
は、製造方法に従って、比表面積、細孔容積を異にする
が、後述する本発明の具体例に沿って使用することによ
り、所定の効果を得ることができるものであれば、それ
らの物性により限定されるものでない。The hydrophobic porous synthetic resin carrier used for adsorbing the partially purified DX may be of any type as long as it has the performance in accordance with the object of the present invention. Examples thereof include a porous (or porous) polymer obtained by polymerizing styrene and divinylbenzene by a special method. Such a porous polymer differs in specific surface area and pore volume according to the production method. However, if it is used in accordance with a specific example of the present invention described later, a predetermined effect can be obtained. It is not limited by their physical properties.
【００１１】しかしながら、一般的に、比表面積が４０
０〜８００ｍ2／ｇの範囲にあり、細孔容積が０.６５０
〜１.５ｍｌ／ｇ にあるものが好適に使用できる。この
ような多孔質ポリマーは、市販されているものから選ぶ
こともでき、限定されるものでないが、三菱化学株式会
社製のダイヤイオン（商標）ＨＰ２０（以下、ＨＰ２０
と略記する）または同ＨＰ２０ＳＳ（以下、ＨＰ２０Ｓ
Ｓと略記する）を都合よく使用することができる。However, in general, the specific surface area is 40
In the range of 0-800 m 2 / g and a pore volume of 0.650.
What is in the range of ~ 1.5 ml / g can be suitably used. Such a porous polymer can be selected from commercially available ones, and is not limited thereto. However, Diaion (trademark) HP20 (hereinafter referred to as HP20) manufactured by Mitsubishi Chemical Corporation.
HP20S (hereinafter abbreviated as HP20S)
S (abbreviated as S) can be conveniently used.
【００１２】上記のような、粗精製ＤＸを吸着させた疎
水性多孔質合成樹脂担体からのＤＸの溶出は、本発明に
よれば、水混和性有機溶媒を含有するｐＨ２.５〜５に
調整された水性溶液を用いて実施される。水混和性有機
溶媒は本発明の目的に沿う限り特に限定されるものでな
いが、経済性と溶出効率の両者を兼ね備えたものとして
は、メタノール、エタノール、イソプロパノールおよび
アセトンを挙げることができる。これらのうち、特に好
適なものは、メタノールおよびアセトンである。このよ
うな溶媒を使用するとき、これらの溶媒と水溶液は、使
用する溶媒によって最適混合比が変動するので限定でき
ないが、一般的に、溶媒：水溶液の比は、それぞれ容量
基準で、１０：９０〜７０：３０の割合で使用できる。
たとえば、メタノールを使用する場合には、メタノー
ル：水溶液の比は、２０：８０〜７０：３０、特に、約
５０：５０の割合で使用でき、アセトンを使用する場合
には、アセトン：水溶液の比は、１０：９０〜３０：７
０、特に１５：８５〜２５：７５の割合で使用できる。According to the present invention, the elution of DX from the hydrophobic porous synthetic resin carrier to which the partially purified DX is adsorbed is adjusted to pH 2.5 to 5 containing a water-miscible organic solvent. It is carried out using the aqueous solution obtained. The water-miscible organic solvent is not particularly limited as long as it meets the purpose of the present invention. Examples of the solvent having both economy and elution efficiency include methanol, ethanol, isopropanol and acetone. Of these, particularly preferred are methanol and acetone. When such a solvent is used, the solvent and the aqueous solution cannot be limited because the optimum mixing ratio varies depending on the solvent to be used. However, in general, the ratio of the solvent to the aqueous solution is 10:90 on a volume basis. It can be used in a ratio of up to 70:30.
For example, when methanol is used, the ratio of methanol: aqueous solution can be 20:80 to 70:30, especially about 50:50. When acetone is used, the ratio of acetone: aqueous solution can be used. Is from 10:90 to 30: 7
It can be used at a ratio of 0, especially 15:85 to 25:75.
【００１３】これらの有機溶媒を含有する水性溶液は、
特にｐＨ２.５〜５に調整されていることが必要であ
る。ｐＨが２.５より低いと、一般的に得られる製品に
おけるＤＸの相対純度が低くなる傾向があり、特にその
後の精製工程で除去しにくい不純物が増加する傾向があ
る。一方、ｐＨが６を超えるとＤＸの溶出に要する溶出
液量が多くなる傾向があり、またＤＸの溶液中での安定
性が低くなる傾向もある。この有機溶媒含有水性溶液の
ｐＨ調整は、無機もしくは有機酸または必要により緩衝
剤を併用して行うことができる。酸としては、特に塩酸
を好ましいものとして挙げることができる。緩衝化に使
用できる緩衝剤は、製品に悪影響を及ぼさないものであ
れば、いかなる緩衝剤であってもよい。限定されるもの
でないが、かような緩衝剤としては、重フタル酸カリウ
ム−ＨＣｌ、グリココール酸−ＨＣｌ、クエン酸ナトリ
ウム−ＨＣｌ、クエン酸カリウム−ＨＣｌ、クエン酸カ
リウム−クエン酸、コハク酸−Ｎａ2Ｂ4Ｏ7、酢酸ナト
リウム−ＨＣｌ、酢酸ナトリウム−酢酸、クエン酸−Ｎ
ａ2ＨＰＯ4、酒石酸−酒石酸ナトリウム、乳酸−乳酸ナ
トリウム、アコニチン酸−ＮａＯＨ、コハク酸−ＮａＯ
Ｈ等を挙げることができる。これらのうち、特に、クエ
ン酸ナトリウム−ＨＣｌが好ましい。Aqueous solutions containing these organic solvents are:
In particular, the pH must be adjusted to 2.5 to 5. When the pH is lower than 2.5, the relative purity of DX in a generally obtained product tends to be low, and in particular, impurities which are difficult to remove in a subsequent purification step tend to increase. On the other hand, when the pH exceeds 6, the amount of eluate required for elution of DX tends to increase, and the stability of DX in the solution tends to decrease. The pH of the aqueous solution containing an organic solvent can be adjusted using an inorganic or organic acid or, if necessary, a buffer. As the acid, particularly preferred is hydrochloric acid. Buffers that can be used for buffering may be any buffers that do not adversely affect the product. Such buffers include, but are not limited to, potassium biphthalate-HCl, glycocholate-HCl, sodium citrate-HCl, potassium citrate-HCl, potassium citrate-citric acid, succinic acid- Na 2 B 4 O 7 , sodium acetate-HCl, sodium acetate-acetic acid, citric acid-N
a 2 HPO 4 , tartaric acid-sodium tartrate, lactic acid-sodium lactate, aconitic acid-NaOH, succinic acid-NaO
H and the like. Of these, sodium citrate-HCl is particularly preferred.
【００１４】塩酸のみを使用してｐＨ調整すると、本操
作以降のＤＸ回収の際にクロロホルム等の有機溶媒によ
る抽出工程等の塩の置換工程を省略することができ、他
の酸の使用により生じる塩または緩衝剤を除去する工程
を省略できる点で有利である。If the pH is adjusted using only hydrochloric acid, a salt replacement step such as an extraction step with an organic solvent such as chloroform can be omitted in the DX recovery after this operation, and the use of another acid will cause the salt to be removed. This is advantageous in that the step of removing the salt or the buffer can be omitted.
【００１５】上記樹脂担体からそれに吸着しているＤＸ
を溶出する操作条件は、ＤＸまたは樹脂担体に変性など
の悪影響を及ぼさない温度であれば、どのような温度で
あってもよいが、ほぼ室温（１０〜３０℃）付近での操
作を選ぶのが都合よい。さらに、かような樹脂担体は、
通常、カラムに充填した態様で操作するのがよいが、こ
れに限定されない。DX adsorbed on the resin carrier
May be used at any temperature as long as it does not adversely affect the DX or the resin carrier such as denaturation, but the operation at approximately room temperature (10 to 30 ° C.) is preferred. Is convenient. Furthermore, such a resin carrier is
Usually, it is preferable to operate in a state of being packed in a column, but it is not limited to this.
【００１６】粗精製ＤＸを吸着させた疎水性多孔質合成
樹脂担体の調製は、上記溶出に悪影響を及ぼさない方法
に従ったものであれば、どのような方法でＤＸが前記樹
脂担体に吸着されたものであってもよい。しかしなが
ら、具体的な調製は、粗精製ドキソルビシンを含むｐＨ
約１〜５に調整された水溶液と前記担体との接触により
その担体にＤＸを吸着させ、次いでｐＨ約１〜５の酸性
水でＤＸ吸着樹脂担体を洗浄することによるのが好まし
い。上記の粗精製ドキソルビシン含有液および酸性水の
ｐＨ調節は、上述したような酸または緩衝剤を使用して
行ってもよいが、好ましくは、塩酸を使用することがで
きる。また、酸性水のｐＨは、約４付近に調節すること
が、特に好ましい。このような酸性水による洗浄は、洗
浄液中に不純物の存在が認められなくなるまで続ける。
この際、酸性水は、通常、樹脂量の２〜６倍量使用され
る。The preparation of the hydrophobic porous synthetic resin carrier to which the partially purified DX is adsorbed is carried out by any method as long as DX is adsorbed on the resin carrier as long as the method does not adversely affect the elution. May be used. However, a specific preparation is the pH with crude doxorubicin.
Preferably, DX is adsorbed on the carrier by contacting the carrier with an aqueous solution adjusted to about 1 to 5 and then washing the DX adsorbing resin carrier with acidic water having a pH of about 1 to 5. The above-mentioned pH adjustment of the partially purified doxorubicin-containing solution and the acidic water may be performed using the above-described acid or buffer, but preferably hydrochloric acid can be used. It is particularly preferable that the pH of the acidic water is adjusted to about 4. Such washing with acidic water is continued until the presence of impurities in the washing liquid is no longer observed.
At this time, the acidic water is usually used in an amount of 2 to 6 times the amount of the resin.
【００１７】以上の本発明方法によって精製されたＤＸ
は、必要により、同一または異なる構成からなる上記方
法で繰り返し処理してもよい。DX purified by the above method of the present invention
May be repeatedly processed by the above-described method having the same or different configuration, if necessary.
【００１８】また、上記の方法により精製されたＤＸ含
有画分からのＤＸの回収は、有機溶媒を留去した後、そ
の溶液から、クロロホルムなどを使用するそれ自体既知
の抽出方法により達成することができる。例えば、相対
純度が一定値以上の画分を集め、その溶液を水酸化ナト
リウムなどでｐＨ約８．５に調節し、溶液量と同量のク
ロロホルム−メタノール混液（５：１）を加えてＤＸを
抽出し、有機溶媒相を減圧下に濃縮した後、これにＤＸ
に対して等モルの塩酸を含むメタノール溶液を加えて塩
酸ＤＸを析出せしめ、析出した塩酸ＤＸを濾取すればよ
い。The recovery of DX from the DX-containing fraction purified by the above method can be achieved by distilling off the organic solvent and then extracting the solution from the solution by using a known extraction method using chloroform or the like. it can. For example, a fraction having a relative purity equal to or higher than a certain value is collected, the solution is adjusted to a pH of about 8.5 with sodium hydroxide or the like, and a chloroform-methanol mixed solution (5: 1) of the same amount as the solution is added to DX Was extracted, and the organic solvent phase was concentrated under reduced pressure.
A hydrochloric acid DX may be precipitated by adding a methanol solution containing an equimolar amount of hydrochloric acid, and the precipitated hydrochloric acid DX may be collected by filtration.
【００１９】塩酸によりｐＨ調整した洗浄液および有機
溶媒含有水性溶液を用いて精製するときには、クロロホ
ルム−メタノール混液などによる抽出工程を省略してＤ
Ｘ含有画分から回収することができる。例えば、相対純
度が一定値以上のＤＸ含有画分を集め、減圧下に濃縮
し、これにアセトンなどの貧溶媒を加えてＤＸを析出せ
しめ、析出したＤＸを濾取すればよい。When purification is carried out using a washing solution adjusted to pH with hydrochloric acid and an aqueous solution containing an organic solvent, the extraction step using a chloroform-methanol mixed solution or the like is omitted and D
It can be recovered from the X-containing fraction. For example, a DX-containing fraction having a relative purity of a certain value or more is collected, concentrated under reduced pressure, a poor solvent such as acetone is added thereto to precipitate DX, and the deposited DX may be collected by filtration.
【００２０】[0020]
【実施例】以下、具体例を挙げて本発明をさらに詳細に
説明するが、これらの例は、本発明ならびに本発明の作
用および効果の理解をより容易にする目的で提供するも
のである。EXAMPLES The present invention will be described in more detail with reference to specific examples, which are provided for the purpose of making the present invention and the operation and effects of the present invention easier to understand.
【００２１】実施例１：低純度ＤＸの精製−クエン酸緩
衝液（以下、ＣＢＳという）（ｐＨ３.５）、溶媒比率
（１５：８５）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ３．５、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水２５ｍｌで洗浄し、続けて
０.１ＭＣＢＳ（ｐＨ３.５）５０ｍｌで洗浄した後、ア
セトン−０.１ＭＣＢＳ（ｐＨ３.５）（１５：８５）で
溶出させた。下記の条件でＤＸ溶出分画（各６．２５ｍ
ｌ）のＨＰＬＣ分析を行い、ＤＸ濃度５００μｇ／ｍｌ
以上の分画を集めた。その結果、回収率５７％、回収溶
液量２２８ｍｌ、相対純度９９．８９％であった。Example 1: Purification of low-purity DX-citrate buffer (hereinafter referred to as CBS) (pH 3.5), solvent ratio (15:85)-500 mg of low-purity DX powder (DX content 397 mg, relative purity 97) .8%) in 25 m of acidic water (pH 3.5, hydrochloric acid)
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 25 ml of the same acidic water and successively with 50 ml of 0.1 MCBS (pH 3.5), elution was carried out with acetone-0.1 MCBS (pH 3.5) (15:85). DX elution fractions (6.25 m each)
1) HPLC analysis, and DX concentration 500 μg / ml
The above fractions were collected. As a result, the recovery rate was 57%, the recovery solution amount was 228 ml, and the relative purity was 99.89%.
【００２２】ＨＰＬＣ分析条件： カラム：ＺＯＲＢＡＸ，ＴＭＳ、 ２５０×４.６ｍｍ
i.d.（ヒューレット・パッカード社製） カラム温度：４０℃ 移動相：水−アセトニトリル−メタノール−リン酸(５
４０：２９０：１７０：２）＋ドデシル硫酸ナトリウム
１．０ｇ／ｌ（ｐＨ３．６、２Ｎ水酸化ナトリウム） 流速：１.５ｍｌ／ｍｉｎ 検出：４９５ｎｍHPLC analysis conditions: Column: ZORBAX, TMS, 250 × 4.6 mm
id (Hewlett-Packard) Column temperature: 40 ° C Mobile phase: water-acetonitrile-methanol-phosphoric acid (5
40: 290: 170: 2) +1.0 g / l sodium dodecyl sulfate (pH 3.6, 2N sodium hydroxide) Flow rate: 1.5 ml / min Detection: 495 nm
【００２３】実施例２：低純度ＤＸの精製−ＣＢＳ（ｐ
Ｈ３.５）、溶媒比率（２０：８０）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ３．５、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水２５ｍｌで洗浄し、続けて
０.１ＭＣＢＳ（ｐＨ３.５）５０ｍｌで洗浄した後、ア
セトン−０.１ＭＣＢＳ（ｐＨ３.５）（２０：８０）で
溶出させ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分
析を行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集め
た。その結果、回収率８３％、回収溶液量１２１ｍｌ、
相対純度９９．９３％であった。Example 2: Purification of low-purity DX-CBS (p
H3.5), solvent ratio (20:80)-500 mg of low-purity DX powder (DX content: 397 mg, relative purity: 97.8%) in 25 m of acidic water (pH 3.5, hydrochloric acid)
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 25 ml of the same acidic water and successively with 50 ml of 0.1 MCBS (pH 3.5), elution was carried out with acetone-0.1 MCBS (pH 3.5) (20:80). The eluted fraction was analyzed by HPLC, and fractions with a DX concentration of 500 μg / ml or more were collected. As a result, the recovery rate was 83%, the recovered solution amount was 121 ml,
The relative purity was 99.93%.
【００２４】実施例３：低純度ＤＸの精製−ＣＢＳ（ｐ
Ｈ３.５）、溶媒比率（２５：７５）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ３．５、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水２５ｍｌで洗浄し、続けて
０.１ＭＣＢＳ（ｐＨ３.０）５０ｍｌで洗浄した後、ア
セトン−０.１ＭＣＢＳ（ｐＨ３.５）（２５：７５）で
溶出させ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分
析を行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集め
た。その結果、回収率９６％、回収溶液量９４ｍｌ、相
対純度９９．９６％であった。Example 3: Purification of low-purity DX-CBS (p
H3.5), solvent ratio (25:75) -500 mg of low-purity DX powder (DX content 397 mg, relative purity 97.8%) in acidic water (pH 3.5, hydrochloric acid) 25 m
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 25 ml of the same acidic water and successively with 50 ml of 0.1 MCBS (pH 3.0), elution was carried out with acetone-0.1 MCBS (pH 3.5) (25:75). The eluted fraction was analyzed by HPLC, and fractions with a DX concentration of 500 μg / ml or more were collected. As a result, the recovery rate was 96%, the recovery solution amount was 94 ml, and the relative purity was 99.96%.
【００２５】実施例４：低純度ＤＸの精製−ＣＢＳ（ｐ
Ｈ３.０）、溶媒比率（２０：８０）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ３．０、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水２５ｍｌで洗浄し、続けて
０.１ＭＣＢＳ（ｐＨ３.０）５０ｍｌで洗浄した後、ア
セトン−０.１ＭＣＢＳ（ｐＨ３.０）（２０：８０）で
溶出させ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分
析を行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集め
た。その結果、回収率８６％、回収溶液量１０１ｍｌ、
相対純度９９．９１％であった。Example 4: Purification of low-purity DX-CBS (p
H3.0), solvent ratio (20:80) -500 mg of low-purity DX powder (DX content 397 mg, relative purity 97.8%) in 25 m of acidic water (pH 3.0, hydrochloric acid)
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 25 ml of the same acidic water and successively with 50 ml of 0.1 MCBS (pH 3.0), elution was carried out with acetone-0.1 MCBS (pH 3.0) (20:80). The eluted fraction was analyzed by HPLC, and fractions with a DX concentration of 500 μg / ml or more were collected. As a result, a recovery rate of 86%, a recovery solution amount of 101 ml,
The relative purity was 99.91%.
【００２６】実施例５：低純度ＤＸの精製−ＣＢＳ（ｐ
Ｈ４.０）、溶媒比率（２０：８０）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ４．０、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水２５ｍｌで洗浄し、続けて
０.１ＭＣＢＳ（ｐＨ４．０）５０ｍｌで洗浄した後、
アセトン−０.１ＭＣＢＳ（ｐＨ４．０）（２０：８
０）で溶出させ、実施例１の条件でＤＸ溶出分画のＨＰ
ＬＣ分析を行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画
を集めた。その結果、回収率８９％、回収溶液量２４１
ｍｌ、相対純度９９．８５％であった。Example 5: Purification of low-purity DX-CBS (p
H4.0), solvent ratio (20:80)-500 mg of low-purity DX powder (DX content: 397 mg, relative purity: 97.8%) in acidic water (pH 4.0, hydrochloric acid) 25 m
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 25 ml of the same acidic water and successively with 50 ml of 0.1 MCBS (pH 4.0),
Acetone-0.1 MCBS (pH 4.0) (20: 8
0) and the HP of the DX eluted fraction under the conditions of Example 1
LC analysis was performed and fractions with a DX concentration of 500 μg / ml or more were collected. As a result, the recovery rate was 89% and the amount of the recovered solution was 241.
ml, relative purity 99.85%.
【００２７】実施例６：低純度ＤＸの精製−塩酸水（ｐ
Ｈ３.０）、溶媒比率（１０：９０）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ３．０、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、ア
セトン−塩酸水（ｐＨ３．０）（１０：９０）で溶出さ
せ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を行
い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。そ
の結果、回収率８９％、回収溶液量６０ｍｌ、相対純度
９９．９２％であった。Example 6: Purification of low-purity DX-aqueous hydrochloric acid (p
H3.0), solvent ratio (10:90) -500 mg of low-purity DX powder (DX content 397 mg, relative purity 97.8%) in 25 m of acidic water (pH 3.0, hydrochloric acid) 25 m
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 50 ml of the same acidic water, elution was carried out with acetone-hydrochloric acid solution (pH 3.0) (10:90), HPLC analysis of the DX eluted fraction was performed under the conditions of Example 1, and a DX concentration of 500 μg / ml or more was obtained. Fractions were collected. As a result, the recovery rate was 89%, the recovered solution volume was 60 ml, and the relative purity was 99.92%.
【００２８】実施例７：低純度ＤＸの精製−塩酸水（ｐ
Ｈ３.０）、溶媒比率（１５：８５）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ３．０、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、ア
セトン−塩酸水（ｐＨ３．０）（１５：８５）で溶出さ
せ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を行
い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。そ
の結果、回収率９９％、回収溶液量３４ｍｌ、相対純度
９９．９４％であった。Example 7: Purification of low-purity DX-hydrochloric acid aqueous solution (p
H3.0), solvent ratio (15:85)-500 mg of low-purity DX powder (DX content 397 mg, relative purity 97.8%) in 25 m of acidic water (pH 3.0, hydrochloric acid)
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 50 ml of the same acidic water, elution was carried out with acetone-hydrochloric acid solution (pH 3.0) (15:85), and HPLC analysis of the DX eluted fraction under the conditions of Example 1 was carried out to obtain a DX concentration of 500 μg / ml or more. Fractions were collected. As a result, the recovery rate was 99%, the recovery solution amount was 34 ml, and the relative purity was 99.94%.
【００２９】実施例８：低純度ＤＸの精製−塩酸水（ｐ
Ｈ３.０）、溶媒比率（２０：８０）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ３．０、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、ア
セトン−塩酸水（ｐＨ３．０）（２０：８０）で溶出さ
せ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を行
い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。そ
の結果、回収率９９％、回収溶液量２５ｍｌ、相対純度
９９．７４％であった。Example 8: Purification of low-purity DX-hydrochloric acid aqueous solution (p
H3.0), solvent ratio (20:80) -500 mg of low-purity DX powder (DX content 397 mg, relative purity 97.8%) in 25 m of acidic water (pH 3.0, hydrochloric acid)
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 50 ml of the same acidic water, elution was carried out with acetone-hydrochloric acid aqueous solution (pH 3.0) (20:80), and HPLC analysis of the DX elution fraction was carried out under the conditions of Example 1, and the DX concentration was 500 μg / ml or more. Fractions were collected. As a result, the recovery rate was 99%, the recovered solution amount was 25 ml, and the relative purity was 99.74%.
【００３０】実施例９：低純度ＤＸの精製−塩酸水（ｐ
Ｈ３.０）、溶媒比率（１０：９０）、負荷量２倍− 低純度ＤＸ粉末１０００ｍｇ（ＤＸ含量７９４ｍｇ、相
対純度９７．８％）を酸性水（ｐＨ３．０、塩酸）２５
ｍｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２
５ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、
アセトン−塩酸水（ｐＨ３．０）（１０：９０）で溶出
させ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を
行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。
その結果、回収率８０％、回収溶液量５４ｍｌ、相対純
度９９．９３％であった。Example 9: Purification of low-purity DX-aqueous hydrochloric acid (p
H3.0), solvent ratio (10:90), loading 2 times-1000 mg of low-purity DX powder (DX content 794 mg, relative purity 97.8%) was added to acidic water (pH 3.0, hydrochloric acid) 25.
The solution was dissolved in a column of HP20SS (2
5 ml). After washing with 50 ml of the same acidic water,
The fraction was eluted with acetone-aqueous hydrochloric acid (pH 3.0) (10:90), and the fraction eluted with DX was analyzed by HPLC under the conditions of Example 1 to obtain a fraction with a DX concentration of 500 μg / ml or more.
As a result, the recovery rate was 80%, the recovered solution amount was 54 ml, and the relative purity was 99.93%.
【００３１】実施例１０：低純度ＤＸの精製−塩酸水
（ｐＨ３.０）、溶媒比率（１５：８５）、負荷量２倍
− 低純度ＤＸ粉末１０００ｍｇ（ＤＸ含量７９４ｍｇ、相
対純度９７．８％）を酸性水（ｐＨ３．０、塩酸）２５
ｍｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２
５ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、
アセトン−塩酸水（ｐＨ３．０）（１５：８５）で溶出
させ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を
行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。
その結果、回収率８１％、回収溶液量３８ｍｌ、相対純
度９９．９１％であった。Example 10: Purification of low-purity DX-hydrochloric acid aqueous solution (pH 3.0), solvent ratio (15:85), double the loading-1000 mg of low-purity DX powder (DX content: 794 mg, relative purity: 97.8%) ) In acidic water (pH 3.0, hydrochloric acid) 25
The solution was dissolved in a column of HP20SS (2
5 ml). After washing with 50 ml of the same acidic water,
The fraction was eluted with acetone-aqueous hydrochloric acid (pH 3.0) (15:85) and subjected to HPLC analysis of the fraction eluted with DX under the conditions of Example 1 to collect fractions having a DX concentration of 500 μg / ml or more.
As a result, the recovery rate was 81%, the recovery solution amount was 38 ml, and the relative purity was 99.91%.
【００３２】実施例１１：低純度ＤＸの精製−塩酸水
（ｐＨ４.０）、溶媒比率（１５：８５）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ４．０、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、ア
セトン−塩酸水（ｐＨ４．０）（１５：８５）で溶出さ
せ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を行
い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。そ
の結果、回収率９８％、回収溶液量４０ｍｌ、相対純度
９９．９６％であった。Example 11: Purification of low-purity DX-hydrochloric acid aqueous solution (pH 4.0), solvent ratio (15:85) -500 mg of low-purity DX powder (DX content 397 mg, relative purity 97.8%) was added to acidic water ( pH 4.0, hydrochloric acid) 25m
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 50 ml of the same acidic water, elution was carried out with acetone-hydrochloric acid solution (pH 4.0) (15:85), HPLC analysis of the DX eluted fraction was performed under the conditions of Example 1, and a DX concentration of 500 μg / ml or more was obtained. Fractions were collected. As a result, the recovery rate was 98%, the recovery solution amount was 40 ml, and the relative purity was 99.96%.
【００３３】実施例１２：低純度ＤＸの精製−塩酸水
（ｐＨ４.０）、溶媒比率（２０：８０）− 低純度ＤＸ粉末５００ｍｇ（ＤＸ含量３９７ｍｇ、相対
純度９７．８％）を酸性水（ｐＨ４．０、塩酸）２５ｍ
ｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２５
ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、ア
セトン−塩酸水（ｐＨ４．０）（２０：８０）で溶出さ
せ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を行
い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。そ
の結果、回収率９９％、回収溶液量２９ｍｌ、相対純度
９９．８１％であった。Example 12: Purification of low-purity DX-hydrochloric acid aqueous solution (pH 4.0), solvent ratio (20:80)-500 mg of low-purity DX powder (DX content 397 mg, relative purity 97.8%) was added to acidic water ( pH 4.0, hydrochloric acid) 25m
1 and dissolve this solution in a column of HP20SS (25
ml). After washing with 50 ml of the same acidic water, elution was carried out with acetone-hydrochloric acid solution (pH 4.0) (20:80), and the HPLC elution fraction was subjected to HPLC analysis under the conditions of Example 1 to obtain a DX concentration of 500 μg / ml or more. Fractions were collected. As a result, the recovery rate was 99%, the recovered solution amount was 29 ml, and the relative purity was 99.81%.
【００３４】実施例１３：低純度ＤＸの精製−塩酸水
（ｐＨ４.０）、溶媒比率（１０：９０）、負荷量２倍
− 低純度ＤＸ粉末１０００ｍｇ（ＤＸ含量７９４ｍｇ、相
対純度９７．８％）を酸性水（ｐＨ４．０、塩酸）２５
ｍｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２
５ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、
アセトン−塩酸水（ｐＨ４．０）（１０：９０）で溶出
させ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を
行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。
その結果、回収率８０％、回収溶液量５４ｍｌ、相対純
度９９．９４％であった。Example 13: Purification of low-purity DX-hydrochloric acid aqueous solution (pH 4.0), solvent ratio (10:90), double loading-1000 mg of low-purity DX powder (DX content: 794 mg, relative purity: 97.8%) ) In acidic water (pH 4.0, hydrochloric acid) 25
The solution was dissolved in a column of HP20SS (2
5 ml). After washing with 50 ml of the same acidic water,
Elution was carried out with acetone-aqueous hydrochloric acid (pH 4.0) (10:90), and HPLC analysis of the DX eluted fraction was carried out under the conditions of Example 1 to collect fractions having a DX concentration of 500 μg / ml or more.
As a result, the recovery rate was 80%, the recovery solution amount was 54 ml, and the relative purity was 99.94%.
【００３５】実施例１４：低純度ＤＸの精製−塩酸水
（ｐＨ４.０）、溶媒比率（１５：８５）、負荷量２倍
− 低純度ＤＸ粉末１０００ｍｇ（ＤＸ含量７９４ｍｇ、相
対純度９７．８％）を酸性水（ｐＨ４．０、塩酸）２５
ｍｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２
５ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、
アセトン−塩酸水（ｐＨ４．０）（１５：８５）で溶出
させ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を
行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。
その結果、回収率８２％、回収溶液量４３ｍｌ、相対純
度９９．９０％であった。Example 14: Purification of low-purity DX-hydrochloric acid aqueous solution (pH 4.0), solvent ratio (15:85), double loading-1000 mg of low-purity DX powder (DX content 794 mg, relative purity 97.8%) ) In acidic water (pH 4.0, hydrochloric acid) 25
The solution was dissolved in a column of HP20SS (2
5 ml). After washing with 50 ml of the same acidic water,
The fraction was eluted with acetone-aqueous hydrochloric acid (pH 4.0) (15:85) and subjected to HPLC analysis of the fraction eluted with DX under the conditions of Example 1 to collect fractions having a DX concentration of 500 μg / ml or more.
As a result, the recovery rate was 82%, the recovered solution amount was 43 ml, and the relative purity was 99.90%.
【００３６】実施例１５：低純度ＤＸの精製−塩酸水
（ｐＨ２.５）、溶媒比率（１５：８５）、負荷量２倍
− 低純度ＤＸ粉末１０００ｍｇ（ＤＸ含量７９４ｍｇ、相
対純度９７．８％）を酸性水（ｐＨ２．５、塩酸）２５
ｍｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２
５ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、
アセトン−塩酸水（ｐＨ２．５）（１５：８５）で溶出
させ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を
行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。
その結果、回収率８５％、回収溶液量３８ｍｌ、相対純
度９９．８３％であった。Example 15: Purification of low-purity DX-hydrochloric acid aqueous solution (pH 2.5), solvent ratio (15:85), double loading-1000 mg of low-purity DX powder (DX content: 794 mg, relative purity: 97.8%) ) In acidic water (pH 2.5, hydrochloric acid) 25
The solution was dissolved in a column of HP20SS (2
5 ml). After washing with 50 ml of the same acidic water,
Elution was carried out with acetone-hydrochloric acid aqueous solution (pH 2.5) (15:85), and HPLC analysis of the DX elution fraction was performed under the conditions of Example 1 to collect a fraction having a DX concentration of 500 μg / ml or more.
As a result, the recovery rate was 85%, the recovery solution amount was 38 ml, and the relative purity was 99.83%.
【００３７】実施例１６：低純度ＤＸの精製−塩酸水
（ｐＨ５.０）、溶媒比率（１５：８５）、負荷量２倍
− 低純度ＤＸ粉末１０００ｍｇ（ＤＸ含量７９４ｍｇ、相
対純度９７．８％）を酸性水（ｐＨ５．０、塩酸）２５
ｍｌに溶解して、この溶液をＨＰ２０ＳＳのカラム（２
５ｍｌ）に通液した。同酸性水５０ｍｌで洗浄した後、
アセトン−塩酸水（ｐＨ５．０）（１５：８５）で溶出
させ、実施例１の条件でＤＸ溶出分画のＨＰＬＣ分析を
行い、ＤＸ濃度５００μｇ／ｍｌ以上の分画を集めた。
その結果、回収率９４％、回収溶液量４９ｍｌ、相対純
度９９．８７％であった。Example 16: Purification of low-purity DX-hydrochloric acid aqueous solution (pH 5.0), solvent ratio (15:85), double the loading-1000 mg of low-purity DX powder (DX content: 794 mg, relative purity: 97.8%) ) In acidic water (pH 5.0, hydrochloric acid) 25
The solution was dissolved in a column of HP20SS (2
5 ml). After washing with 50 ml of the same acidic water,
The fraction was eluted with acetone-aqueous hydrochloric acid (pH 5.0) (15:85), and the DX-eluted fraction was subjected to HPLC analysis under the conditions of Example 1 to collect fractions having a DX concentration of 500 μg / ml or more.
As a result, the recovery rate was 94%, the amount of the recovered solution was 49 ml, and the relative purity was 99.87%.
【００３８】実施例１７：ＤＸ反応液の精製−塩酸水
（ｐＨ３.０）、溶媒比率（１０：９０）− １４−ブロモドキソルビシン１０００ｍｇを水１７ｍｌ
に溶解した。これにギ酸ナトリウム水溶液（ギ酸ナトリ
ウム３３８ｍｇ／水３ｍｌ、ｐＨ５．９）を加え、窒素
置換しながら２０時間攪拌した。得られた反応終了液
（ｐＨ４）に０．１Ｎ塩酸３０ｍｌおよび酸性水（ｐＨ
３．０、塩酸）を加えた（液量７５ｍｌ、ＤＸ含量約６
５０ｍｇ）。この溶液をＨＰ２０ＳＳのカラム（２５ｍ
ｌ）に通液した。酸性水（ｐＨ３．０、塩酸）５０ｍｌ
で洗浄した後、アセトン−塩酸水（ｐＨ３．０）（１
０：９０）で溶出させ、実施例１の条件でＤＸ溶出分画
のＨＰＬＣ分析を行い、ＤＸ濃度５００μｇ／ｍｌ以上
の分画を集めた。その結果、回収率７８％、回収溶液量
５４ｍｌ、相対純度９８．８９％であった。Example 17: Purification of DX reaction solution-hydrochloric acid aqueous solution (pH 3.0), solvent ratio (10:90)-1000 mg of 14-bromodoxorubicin in 17 ml of water
Was dissolved. To this was added an aqueous solution of sodium formate (338 mg of sodium formate / 3 ml of water, pH 5.9), and the mixture was stirred for 20 hours while purging with nitrogen. 30 ml of 0.1N hydrochloric acid and acidic water (pH
3.0, hydrochloric acid) (liquid volume 75 ml, DX content about 6).
50 mg). This solution was applied to a column of HP20SS (25 m
1). 50 ml of acidic water (pH 3.0, hydrochloric acid)
After washing with acetone, an aqueous solution of acetone-hydrochloric acid (pH 3.0) (1
0:90), and the DX-eluted fraction was analyzed by HPLC under the conditions of Example 1 to collect fractions with a DX concentration of 500 μg / ml or more. As a result, the recovery rate was 78%, the recovery solution amount was 54 ml, and the relative purity was 98.89%.
【００３９】実施例１８：ＤＸ反応液の精製−塩酸水
（ｐＨ３.０）、溶媒比率（１５：８５）− 実施例１７と同様に調製したドキソルビシン含有反応終
了液（ｐＨ４）に０．１Ｎ塩酸３０ｍｌおよび酸性水
（ｐＨ３．０、塩酸）を加えた（液量７５ｍｌ、ＤＸ含
量約６５０ｍｇ）。この溶液をＨＰ２０ＳＳのカラム
（２５ｍｌ）に通液した。酸性水（ｐＨ３．０、塩酸）
５０ｍｌで洗浄した後、アセトン−塩酸水（ｐＨ３．
０）（１５：８５）で溶出させ、実施例１の条件でＤＸ
溶出分画のＨＰＬＣ分析を行い、ＤＸ濃度５００μｇ／
ｍｌ以上の分画を集めた。その結果、回収率８８％、回
収溶液量５４ｍｌ、相対純度９９．９１％であった。Example 18: Purification of DX reaction solution-hydrochloric acid aqueous solution (pH 3.0), solvent ratio (15:85)-0.1 N hydrochloric acid was added to the doxorubicin-containing reaction end solution (pH 4) prepared in the same manner as in Example 17. 30 ml and acidic water (pH 3.0, hydrochloric acid) were added (liquid volume 75 ml, DX content about 650 mg). This solution was passed through a column (25 ml) of HP20SS. Acidic water (pH 3.0, hydrochloric acid)
After washing with 50 ml, acetone-hydrochloric acid solution (pH 3.
0) (15:85) and DX under the conditions of Example 1.
The eluted fraction was analyzed by HPLC, and the DX concentration was 500 μg /
More than ml fractions were collected. As a result, the recovery rate was 88%, the recovery solution amount was 54 ml, and the relative purity was 99.91%.
Claims (9)
ンを吸着させた疎水性多孔質合成樹脂担体を、水混和性
有機溶媒を含有するｐＨ２.５〜５に調整された水性溶
液による溶出操作にかけることによりドキソルビシンを
溶出し、こうして得られた溶出液から精製されたドキソ
ルビシンを回収することを特徴とするドキソルビシンの
精製方法。1. Doxorubicin obtained by subjecting a hydrophobic porous synthetic resin carrier obtained by adsorbing doxorubicin from partially purified doxorubicin to an elution operation with an aqueous solution containing a water-miscible organic solvent and adjusted to pH 2.5 to 5. And a method of purifying doxorubicin, comprising recovering purified doxorubicin from the eluate thus obtained.
性多孔質合成樹脂担体が、粗精製ドキソルビシンを含む
ｐＨ約１〜５に調整された水溶液と疎水性多孔質合成樹
脂担体との接触により前記担体にドキソルビシンを吸着
させ、次いでｐＨ約１〜５の酸性水溶液でドキソルビシ
ンを吸着した前記担体を洗浄することにより調製される
請求項１記載の精製方法。2. The method according to claim 1, wherein the hydrophobic porous synthetic resin carrier to which the roughly purified doxorubicin is adsorbed is contacted with an aqueous solution containing the roughly purified doxorubicin and adjusted to a pH of about 1 to 5 and the hydrophobic porous synthetic resin carrier. 2. The purification method according to claim 1, wherein the doxorubicin is adsorbed on the carrier, and the carrier having adsorbed doxorubicin is washed with an acidic aqueous solution having a pH of about 1 to 5.
５である請求項２記載の精製方法。3. The pH of the acidic aqueous solution is about 2.5-3.
3. The purification method according to claim 2, which is 5.
浄するのに用いられる前記酸性水溶液のｐＨが塩酸によ
り調整されたものである請求項２または３記載の精製方
法。4. The purification method according to claim 2, wherein the pH of the acidic aqueous solution used for washing the carrier having adsorbed doxorubicin is adjusted with hydrochloric acid.
が塩酸により調整されており、そして前記有機溶媒がア
セトンである請求項１〜４のいずれかに記載の精製方
法。5. The pH of an aqueous solution containing the organic solvent
The purification method according to any one of claims 1 to 4, wherein is adjusted with hydrochloric acid, and the organic solvent is acetone.
セトン：水溶液の容量比が１０：９０〜３０：７０の範
囲にある請求項５記載の精製方法。6. The purification method according to claim 5, wherein the aqueous solution containing acetone has a volume ratio of acetone: aqueous solution in the range of 10:90 to 30:70.
が塩酸により調整されており、そして前記有機溶媒がメ
タノールである請求項１〜４のいずれかに記載の精製方
法。7. The pH of an aqueous solution containing the organic solvent
The purification method according to any one of claims 1 to 4, wherein is adjusted with hydrochloric acid, and the organic solvent is methanol.
タノール：水溶液の容量比が２０：８０〜７０：３０の
範囲にある請求項７記載の精製方法。8. The purification method according to claim 7, wherein the aqueous solution containing the organic solvent has a methanol: aqueous solution volume ratio in the range of 20:80 to 70:30.
〜９８％を有する請求項１〜８のいずれかに記載の精製
方法。9. The crude doxorubicin has a relative purity of 90%.
The purification method according to any one of claims 1 to 8, wherein the purification method has about 98%.
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|---|---|---|---|
| JP11161030A JP2000351791A (en) | 1999-06-08 | 1999-06-08 | Purification of doxorubicin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11161030A JP2000351791A (en) | 1999-06-08 | 1999-06-08 | Purification of doxorubicin |
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| Publication Number | Publication Date |
|---|---|
| JP2000351791A true JP2000351791A (en) | 2000-12-19 |
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ID=15727273
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100351718B1 (en) * | 1999-11-02 | 2002-09-11 | 보령제약 주식회사 | A purifying process of 4'-epi-doxorubicin |
| CN104774229A (en) * | 2014-01-10 | 2015-07-15 | 浙江华谱新创科技有限公司 | Preparation method for doxorubicin and derivative thereof |
| WO2017075994A1 (en) * | 2015-11-05 | 2017-05-11 | 浙江海正药业股份有限公司 | Separation and purification method for epirubicin or hydrochloride thereof |
| CN111574574A (en) * | 2020-04-20 | 2020-08-25 | 苏州赛分科技有限公司 | Method for purifying doxorubicin |
| US20210308358A1 (en) * | 2016-05-31 | 2021-10-07 | Penumbra, Inc. | Methods and apparatus for extracting doxorubicin from blood and measuring doxorubicin in blood |
-
1999
- 1999-06-08 JP JP11161030A patent/JP2000351791A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100351718B1 (en) * | 1999-11-02 | 2002-09-11 | 보령제약 주식회사 | A purifying process of 4'-epi-doxorubicin |
| CN104774229A (en) * | 2014-01-10 | 2015-07-15 | 浙江华谱新创科技有限公司 | Preparation method for doxorubicin and derivative thereof |
| WO2017075994A1 (en) * | 2015-11-05 | 2017-05-11 | 浙江海正药业股份有限公司 | Separation and purification method for epirubicin or hydrochloride thereof |
| CN108350015A (en) * | 2015-11-05 | 2018-07-31 | 浙江海正药业股份有限公司 | A kind of isolation and purification method of epirubicin or its hydrochloride |
| US20210308358A1 (en) * | 2016-05-31 | 2021-10-07 | Penumbra, Inc. | Methods and apparatus for extracting doxorubicin from blood and measuring doxorubicin in blood |
| CN111574574A (en) * | 2020-04-20 | 2020-08-25 | 苏州赛分科技有限公司 | Method for purifying doxorubicin |
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