JP4031380B2 - Method for analyzing platinum-containing compounds - Google Patents

Description

で示されるジクロロ[(1R,2R)-1,2-シクロヘキサンジアミン-N,N']プラチナム（Dichloro[(1R,2R)-1,2-cyclohexanediamine-N,N']platinum）（以下、化合物（A）と記すこともある。）、式化２
【化２】

で示されるシス[(1R,2R)-(1,2-シクロヘキサンジアミン-N,N')ビス(ミリスタート)]プラチナム(ＩＩ)（cis[(1R,2R)-(1,2-cyclohexanediamine-N,N')bis(myristato)]platinum(ＩＩ)）（以下、化合物（Ｂ）と記すこともある。）等の[(1R,2R)-1,2-シクロヘキサンジアミン-N,N']プラチナム系化合物、さらに例えば、式化３から式化５
【化３】

【化４】

【化５】

で示される白金元素にシステイン、メチオニン等のアミノ酸が配位してなる白金含有化合物が挙げられる。
【０００６】
生体試料としては、哺乳動物から採取された生体成分全般が挙げられ、例えば、血清、血漿、尿、組織等の生体成分を挙げることができる。
生体試料は、哺乳動物から通常の方法により入手される。例えば、血清、血漿の場合には哺乳動物から得られた血液を、必要に応じてヘパリン処理した後、遠心分離することによりそれぞれ分離した成分として得られる。また、尿、組織の場合は採取、切除等の方法によって得られる。
【０００７】
本発明方法において第一工程における生体試料を高速液体クロマトグラフィー（HPLC）に供するための前処理としては、例えば、
（1）血清又は血漿を有機溶媒で抽出することにより、抽出上清を一次分析試料として得る方法、（2）血清を限外濾過することにより、限外濾過液を一次分析試料として得る方法、（３）組織に水を添加して得られた混合物をホモジナイズした後、得られたホモジネート又は尿を有機溶媒で抽出することにより、抽出上清を一次分析試料として得る方法、（４）尿を固相抽出することにより、抽出液を一次分析試料として得る方法等が挙げられる。
それぞれの生体成分からの抽出に用いられる有機溶媒としては、例えばメタノール、酢酸エチル、アセトニトリル等が挙げられる。
【０００８】
次いで、第一工程における前処理を施すこと又は白金含有化合物を含有する生体試料をそのまま用いることによって調製された一次分析試料は、第二工程における高速液体クロマトグラフィーに供される。生体試料が尿の場合には、採取されたそのままの尿を一次分析試料として第二工程における高速液体クロマトグラフィーに供してもよい。
【０００９】
本発明方法における第二工程では、
固定相から移動相への白金含有化合物の溶解度の差が制御される逆相カラムであって、有機溶媒−水系のイオンペア試薬添加移動相の溶出力が有機溶媒の混合比を勾配的に増加させることにより制御される逆相カラムを用いる高速液体クロマトグラフィーが使用される。
【００１０】
第二工程において用いられる有機溶媒−水系のイオンペア試薬添加移動相において、有機溶媒−水系の移動相としては、通常、逆相カラムを用いた高速液体クロマトグラフィーで使用される有機溶媒及び水系の溶液全般を用いればよい。例えば有機溶媒としては、アセトニトリル、エタノール、テトラヒドロフラン、2−プロパノール、メタノール等を挙げることができ、また、水系の溶液としては、水、リン酸ナトリウム緩衝溶液、酢酸ナトリウム緩衝溶液、酢酸アンモニウム緩衝溶液等が挙げられる。
この場合、当該移動相中の有機溶媒の混合比（有機溶媒／水系）を勾配的に増加させることにより、カラムに保持された成分が順に溶出される。
【００１１】
第二工程において用いられる有機溶媒−水系のイオンペア試薬添加移動相において、イオンペア試薬としては、陽イオン分析用イオンペア試薬、陰イオン分析用イオンペア試薬等を挙げることができる。好ましくは、陽イオン分析用イオンペア試薬が挙げられる。
陽イオン分析用イオンペア試薬としては、例えば炭素原子数３〜２０個であるアルキル基を有するスルホン酸塩又は硫酸塩等を挙げることができる。具体的には、ペンタンスルホン酸ナトリウム、ヘキサンスルホン酸ナトリウム、ヘプタンスルホン酸ナトリウム、オクタンスルホン酸ナトリウム、ドデカンスルホン酸ナトリウム、ドデシル硫酸ナトリウム等が挙げられる。
【００１２】
第二工程における逆相カラムを用いる高速液体クロマトグラフィーの条件は次のとおりである。
移動相中のイオンペア試薬の濃度は、通常、約5〜20mM程度である。有機溶媒の混合比（即ち、移動相全体に対する有機溶媒の混合割合）は、0〜100%であり、その混合割合は分離する化合物に応じて、適宜設定される。逆相カラムには、シリカにオクタデシル基、オクチル基等を化学結合させた固定相が充填された逆相カラムが用いられ、当該カラムの内径は、通常、約2〜5mmI.D.程度、長さは、通常、約50〜250mm程度である。移動相の流速は、通常、約0.1〜2mL/min.程度である。インジェクション量は、カラム径に応じて、適宜設定すればよい。
【００１３】
本発明方法における第三工程では、第二工程により分離された二次分析試料を、酸素が１％（v/v）〜２０％（v/v）の割合で混合されたアルゴンガスをキャリアガスとして用い、かつ誘導結合プラズマ法によるイオン化手段を用いる質量分析（ＩＣＰ-ＭＳ）に供すればよい。
【００１４】
第三工程における誘導結合プラズマ法によるイオン化手段を用いる質量分析（ICP-MS）において、元素をイオン化する方法として、例えば、アルゴンプラズマを発生させる方法等を挙げることができる。
【００１５】
上記の誘導結合プラズマ法によるイオン化手段を用いる質量分析（ICP-MS）の条件は次のとおりである。
酸素が１％（v/v）〜２０％（v/v）、好ましくは３％（v/v）〜１５％（v/v）の割合で混合されたアルゴンガスがキャリアガスとして用いられる。
キャリアガスは、ICP−MS装置への導入前にアルゴンガスと酸素ガスとを混合することにより調製される。
【００１６】
二次分析試料に含有される白金含有化合物を、二次分析試料を誘導結合プラズマ法によるイオン化手段を用いる質量分析方法（ICP-MS）に供することによって白金元素に由来するｍ/ｚ＝194,195,196を検出し、得られたピークの積分値を検量線と比較することにより白金量が定量される。
【００１７】
【実施例】
以下、本発明を実施例により更に詳細に説明するが、本発明は実施例に限定されるものではない。
分析条件
＜ＨＰＬＣ条件＞
移動相：１ｍＭヘプタンスルホン酸ナトリウム水溶液とメタノールとのグラジエント
（１）グラジエント条件Ａ（実施例１で使用）
0 min. 100/0, 10 min. 100/0, 20 min. 0/100, 35 min. 0/100,
36 min. 100/0, 50 min. 100/0（１ｍＭヘプタンスルホン酸ナトリウム水溶液／メタノール）
（２）グラジエント条件B (実施例2で使用)；
0 min. 100/0, 10 min. 100/0, 20 min. 80/20, 25 min. 75/25,
38 min. 70/30, 40 min. 56/44, 48min. 56/44, 62 min. 50/50, 64 min. 0/100, 90 min. 0/100（１ｍＭヘプタンスルホン酸ナトリウム水溶液／メタノール）
検出： ICP-MS（m/z=195（Pt））
カラム：ＯＤＳカラム
＜ICP-MS条件＞
ICP-MS：Agilent4500（Agilent社製）
プラズマ出力： 1.56 〜 1.60 kW
サンプリング位置： 7.5 〜 8.5 mm
アルゴンガス流量
プラズマガス： 約 16 L/min
補助ガス ： 約 1.0 L/min
キャリアガス： 約 0.9 〜 1.1 L/min
キャリアガス中の酸素ガス導入量：５％（Ｖ/Ｖ）〜１０％（Ｖ/Ｖ）
ペリスタリックポンプ回転速度： 0.1 rps（ドレイン側のみ使用）
スプレーチャンバー（S/C）の温度 ：1〜2 ℃
通常導入ネブライザー（Sample Uptake約1.2 mL/min）
プラズマ点灯から測定開始までの安定化時間：40分以上
【００１８】
実施例１ （生体試料中に含まれる化合物（Ａ）の分析例：検出下限及び定量下限の算出）
白金含有化合物が投与されていないラット（即ち、ブランクラット）の血清（Crj:CD(SD)系雄性ラットの7週齢プール血清（オリエンタル酵母工業社製）をセントリフリーYM-30 （アミコン社製）に採取した後、4℃、2000×gで20分間遠心分離（HIMAC CR20B3、日立製作所社製）することにより、限外濾過液を得た。
得られた限外濾過液に化合物（Ａ）を、0、0.5、1、5、10、30及び50ng/mL（化合物（Ａ）の濃度で表示）の濃度となるように添加した。これ（１００μｌ）を上記のＨＰＬＣ条件に従った高速液体クロマトグラフィーに供することによって、二次分析試料を分離し、更に分離された二次分析試料を上記のＩＣＰ−ＭＳ条件に従って誘導結合プラズマ法によるイオン化手段を用いる質量分析に供することによって、当該二次分析試料中に含まれる白金含有化合物を白金元素として元素特異的に検出した。得られたチャート図を図２及び図３に示す。
得られたチャート図において化合物（Ａ）に相当するピークの積分値を用いて、検量線を作成した。その結果を図1に示す。図１から明らかなように0.5ng/mL〜50ng/mLの濃度範囲において直線性が確認された。定量下限は0.025ng of Ptであり、また、ノイズから計算した検出下限は0.0025ng of Ptであった。尚、検出下限及び定量下限は、以下のようにして求められた。
＜定量下限及び検出下限の算定方法＞
検出下限：検出下限をベースラインノイズ（N）に対して有意な差があるピークのシグナル（S）と定義し、ノイズの3倍の強度を持つシグナル（S=3N）を検出下限として次のように算出した。
図2（0.5ng eq. of化合物（Ａ）/mL、試料量100μL、0.025ng eq. of Pt）ではベースラインノイズが約4アバンダンスであり、化合物（Ａ）のシグナル強度が約108アバンダンスであることから、検出下限は、4×3×0.5(ng eq. of 化合物（Ａ）/mL)/108＝0.05ng eq. of 化合物（Ａ）/mL（試料量100μL、0.0025ng eq. of Pt）である。
上記0.05ng eq. of 化合物（Ａ）/mLを測定に用いたとき、検出器に導入された溶液全量中の化合物（Ａ）の量は0.05ng eq. of 化合物（Ａ）/mL×0.1mL=0.005ng eq. of 化合物（Ａ）であり、白金元素の量に換算すると0.005ng eq. of 化合物（Ａ）=0.005×195/380=0.0025ng eq. of Ptとなる。従って、検出下限は白金元素の量に換算して0.0025ngとなる。
定量下限：図2での定量下限値は、化合物（Ａ）の濃度として0.5ng/mL（試料量100μL）である。このとき検出器に導入した溶液全量中の化合物（Ａ）の量は0.5ng eq. of 化合物（Ａ）/mL×0.1mL=0.05ng eq. of 化合物（Ａ）であり、白金元素の量に換算すると0.05ng eq. of 化合物（Ａ）=0.05×195/380=0.025ng eq. of Ptとなる。従って、定量下限は白金元素の量として0.025ngとなる。
【００１９】
実施例２ （生体試料中に含まれる化合物（Ｂ）の分析例）
化合物（Ｂ）の凍結乾燥製剤（住友製薬株式会社社製）に、リピオドールを20mg/mLの濃度になるように添加して懸濁した。得られた懸濁液0.4mLに対して、ラット血清、イヌ血清又はヒト血清4mLを添加した後、得られた混合物を5日間、37℃、5rpmで回転振とうした。５日間後、4℃、3000 rpmで10分間遠心分離（HIMAC CR5DL及びHIMAC CF8DL、日立製作所社製）することにより、リピオドールと血清とを分離した。分離された血清は、直ちにセントリフリーYM-30 （アミコン社製）に採取した後、4℃、2000×gで20分間遠心分離（HIMAC CR20B3、日立製作所社製）することにより、限外濾過液を得た。得られた限外濾過液100uLを、一次分析試料として、実施例１と同様な条件及び方法により、生体試料中に含まれる白金含有化合物を超高感度で分析した。
尚、ラット血清はCrj:CD(SD)系雄性ラットの7週齢プール血清（オリエンタル酵母工業社製）を、イヌ血清は雄性ビーグル犬血清（オリエンタル酵母工業社製）を、ヒト血清は精度管理用凍結乾燥プール血清 登録商標 コンセーラ （日水製薬社製）を使用した。
分析の結果、化合物（Ａ）のメチオニン配位体（即ち、白金元素にメチオニンが配位してなる白金含有化合物）に相当するピークが検出され、その検出プロフィールを図4に示す。
【００２０】
実施例3 （生体試料中に含まれる化合物（Ｂ）の分析例）
化合物（Ｂ）の凍結乾燥製剤（住友製薬株式会社製）にリピオドールを2.5mg/mLの濃度になるように添加して懸濁した。得られた懸濁液をラット（Crj:CD(SD)系雄性ラット7週齢；（日本チャ−ルス・リバ−社製））に50mg/kg単回皮下投与した。
投与後2、6及び24時間目に、ラットをエ−テル麻酔させ、そして当該ラットの後大静脈より全血液を採取した。また投与後24時間までの尿も採取した。血液は氷冷下で約30分間放置した後、4℃、3000 rpmで10分間遠心分離（HIMAC CR5DL及びHIMAC CF8DL、日立製作所社製）することにより、血清を得た。得られた血清は、直ちにセントリフリーYM-30 （アミコン社製）に採取した後、4℃、2000×gで、20分間遠心分離（HIMAC CR20B3、日立製作所社製）することにより、限外濾過液を得た。得られた限外濾過液及び尿200uLを一次分析試料として下記の条件以外は実施例１と同様な条件及び方法により、生体試料中に含まれる白金含有化合物を超高感度で分析した。
分析の結果、化合物（Ａ）のメチオニン配位体（即ち、白金元素にメチオニンが配位してなる白金含有化合物）に相当するピークが検出され、その検出プロフィールを図５及び図６に示す。
＜分析条件＞
移動相：1mMヘプタンスルホン酸ナトリウム水溶液とメタノールとのグラジエント（0/100(v/v)〜100/0(v/v)）
カラム：ODSカラム
＜ＩＣＰ−ＭＳ条件＞
ＩＣＰ−ＭＳ：Agilent４５００（Agilent社製）
プラズマ出力：1.2〜1.6kW
サンプリング位置：0.75〜12.0mm
キャリアガス中の酸素ガス導入量：５％（Ｖ/Ｖ）〜１０％（Ｖ/Ｖ）
キャリアガス流量：0.75〜1.10L/min.
定量下限は0.025ng of Ptであった。
【００２１】
【発明の効果】
本発明により、放射性標識化合物を取り扱う特別な設備や装置を必要とせず、熟練者による高度な操作も必要とされることなく、生体試料中に含まれる白金含有化合物が超高感度で分析可能となった。
【図面の簡単な説明】
【図１】(a) 化合物（Ａ）添加検量線（0.5〜10 ng/mL）。
(b) 化合物（Ａ）添加検量線（0.5〜50 ng/mL）。
【図２】化合物（Ａ）添加検量線（0.5〜10ng/mL）。
【図３】化合物（Ａ）添加検量線（10〜50ng/mL）。
【図４】化合物（Ｂ）のin vitro反応生成物のLC-ICP-MS測定結果（ラット）。
【図５】化合物（Ｂ）投与後ラット血清限外濾過液のLC-ICP-MS測定結果（投与後2時間）。
【図６】化合物（Ｂ）投与後のラットのLC-ICP-MS測定結果（投与後24hr）。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for analyzing a platinum-containing compound contained in a biological sample.
[0002]
[Prior art and problems to be solved by the invention]
Detection methods used in connection with high performance liquid chromatography (HPLC) include ultraviolet absorption photometer (UV), differential refractometer (RI), fluorometer (FP), electrochemical detector (ECD), infrared An absorptiometer (IR) or the like is known, but all of them have a high lower limit of quantification, and specificity in elemental analysis and specificity in chemical form analysis are poor. In addition, as a detection method with a lower limit of quantification and specificity in elemental analysis compared to these detection methods, mass spectrometry using an ionization means by inductively coupled plasma method (ICP-MS: hereinafter, inductively coupled plasma) (Also referred to as mass spectrometry) is known (see Patent Documents 1 and 2). However, when analyzing a platinum-containing compound contained in a biological sample, the biological sample itself is a complex mixture, and therefore substances that interfere with the separation and measurement of the analysis components coexist or are detected by the detector. There is a problem that it is difficult to obtain sufficient sensitivity due to the reason that only a small amount of analysis component is contained. For example, it is known that platinum-containing compounds contained in biological samples are generally analyzed under simple conditions using an inductively coupled plasma mass spectrometer (ICP-MS) connected to high-performance liquid chromatography using a reversed-phase column. However, the lower limit of quantification is about 0.5 ng as the amount of platinum, and sufficient sensitivity has not yet been obtained as a method for analyzing a trace amount of platinum-containing compounds (see Non-Patent Document 1).
On the other hand, the radiolabeled platinum-containing compound is analyzed with a radioactivity measuring apparatus connected to high performance liquid chromatography using a reverse phase column with high sensitivity exceeding the above lower limit of quantification (about 0.5 ng) (quantification) A method that enables analysis at a lower limit of about 0.25 ng is also known, but a special facility and apparatus for handling radiolabeled compounds are required, and advanced operations by experts are required. For these reasons, a method for detecting a platinum-containing compound from a biological sample more easily and with high sensitivity has been demanded.
[0003]
[Patent Document 1]
JP-A-6-242075 (page 2, line 27)
[Patent Document 2]
Japanese Patent Publication No. 2001-114520 (page 4, line 32)
[Non-Patent Document 1]
EARLY BIOTRANSFORMATIONS OF OXALIPLATIN AFTER ITS INTRAVENOUS ADMINISTRATION TO CANCER PATIENTS, Drug Metabolism and Disposition, 28, 1379-1384 (2000) P.1379〜1380
[0004]
[Means for Solving the Problems]
In the method for analyzing a platinum-containing compound in a biological sample, the present inventors performed high-performance liquid chromatography using a reversed-phase column and inductively coupled plasma mass spectrometry, which is a means having excellent specificity in elemental analysis. Use of an ion pair reagent for the mobile phase of high performance liquid chromatography, use of argon gas mixed with oxygen in a ratio of 1% (v / v) to 20% (v / v) as a carrier gas, etc. The present inventors have found that platinum-containing compounds contained in a biological sample can be analyzed with extremely high sensitivity by combining them in the above order under conditions.
That is, the present invention
1. A method for analyzing a platinum-containing compound contained in a biological sample,
(1) By subjecting a biological sample containing a platinum-containing compound to pretreatment for use in high performance liquid chromatography (HPLC) using a reverse phase column or by directly using a biological sample containing a platinum-containing compound, A first step of preparing an analytical sample,
(2) The primary analysis sample prepared in the first step is a reversed-phase column in which the difference in solubility of the platinum-containing compound from the stationary phase to the mobile phase is controlled, and the mobile phase is added with an organic solvent-water ion pair reagent. A second step of separating the secondary analysis sample by subjecting it to high performance liquid chromatography using a reverse phase column in which the elution power of is controlled by gradually increasing the mixing ratio of the organic solvent,
(3) The secondary analysis sample separated in the second step uses an argon gas mixed with oxygen in a ratio of 1% (v / v) to 20% (v / v) as a carrier gas, and is inductively coupled. A third step of element-specific detection of a platinum-containing compound contained in the secondary analysis sample as a platinum element by subjecting it to mass spectrometry (ICP-MS) using ionization means by a plasma method,
(Hereinafter, referred to as the method of the present invention);
2. The platinum-containing compound contained in the biological sample is a [(1R, 2R) -1,2-cyclohexanediamine-N, N '] platinum compound or a platinum-containing metabolite of the compound in vivo. And the method according to item 1 above;
3. 3. The method according to item 2 above, wherein the platinum-containing metabolite in the living body is a platinum-containing compound obtained by coordinating amino acid to platinum element;
4). 3. The method according to item 1 or 2 above, wherein the biological sample is serum, plasma, urine, or tissue collected from a mammal;
5. 5. The method according to item 1, 2, 3 or 4 above, wherein the ion pair reagent used in the second step is an ion pair reagent for cation analysis; 6. The method according to item 5 above, wherein the ion pair reagent for cation analysis is a sulfonate or sulfate having an alkyl group having 3 to 20 carbon atoms;
Etc. are provided.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
In the method of the present invention, examples of the platinum-containing compound contained in the biological sample include [(1R, 2R) -1,2-cyclohexanediamine-N, N ′] platinum compound or platinum in the living body of the compound. Examples thereof include metabolites. More specifically, for example, Formula 1
[Chemical 1]

Dichloro [(1R, 2R) -1,2-cyclohexanediamine-N, N '] platinum (hereinafter referred to as compound) (A) may also be described.), Formula 2
[Chemical 2]

Cis [(1R, 2R)-(1,2-cyclohexanediamine-N, N ′) bis (millistart)] platinum (II) (cis [(1R, 2R)-(1,2-cyclohexanediamine- [(1R, 2R) -1,2-cyclohexanediamine-N, N ′] such as N, N ′) bis (myristato)] platinum (II)) (hereinafter sometimes referred to as compound (B)) Platinum compounds, for example, Formula 3 to Formula 5
[Chemical 3]

[Formula 4]

[Chemical formula 5]

And a platinum-containing compound in which an amino acid such as cysteine or methionine is coordinated to the platinum element represented by
[0006]
Examples of the biological sample include all biological components collected from mammals, and examples thereof include biological components such as serum, plasma, urine, and tissue.
The biological sample is obtained from a mammal by a usual method. For example, in the case of serum and plasma, blood obtained from a mammal is obtained as a separated component by subjecting it to heparin treatment as necessary and then centrifuging it. In the case of urine or tissue, it can be obtained by methods such as collection and excision.
[0007]
In the method of the present invention, as a pretreatment for subjecting the biological sample in the first step to high performance liquid chromatography (HPLC), for example,
(1) A method of obtaining an extraction supernatant as a primary analysis sample by extracting serum or plasma with an organic solvent, (2) A method of obtaining an ultrafiltrate as a primary analysis sample by ultrafiltration of serum, (3) A method of obtaining an extraction supernatant as a primary analysis sample by homogenizing a mixture obtained by adding water to a tissue and then extracting the obtained homogenate or urine with an organic solvent, (4) urine Examples include a method of obtaining an extract as a primary analysis sample by solid phase extraction.
Examples of the organic solvent used for extraction from each biological component include methanol, ethyl acetate, acetonitrile, and the like.
[0008]
Next, the primary analysis sample prepared by performing the pretreatment in the first step or using the biological sample containing the platinum-containing compound as it is is subjected to high performance liquid chromatography in the second step. When the biological sample is urine, the collected urine may be used as a primary analysis sample for high performance liquid chromatography in the second step.
[0009]
In the second step in the method of the present invention,
A reversed-phase column in which the difference in solubility of the platinum-containing compound from the stationary phase to the mobile phase is controlled, and the dissolution power of the organic solvent-water ion pair reagent-added mobile phase increases the organic solvent mixing ratio in a gradient manner. High performance liquid chromatography using a reverse phase column controlled by the process is used.
[0010]
In the organic solvent-water ion pair reagent-added mobile phase used in the second step, the organic solvent-water mobile phase is usually an organic solvent and an aqueous solution used in high performance liquid chromatography using a reverse phase column. General can be used. For example, examples of the organic solvent include acetonitrile, ethanol, tetrahydrofuran, 2-propanol, methanol and the like, and examples of the aqueous solution include water, sodium phosphate buffer solution, sodium acetate buffer solution, ammonium acetate buffer solution and the like. Is mentioned.
In this case, the components retained in the column are eluted in order by increasing the mixing ratio (organic solvent / water system) of the organic solvent in the mobile phase in a gradient manner.
[0011]
In the organic solvent-water ion pair reagent-added mobile phase used in the second step, examples of the ion pair reagent include an ion pair reagent for cation analysis and an ion pair reagent for anion analysis. Preferably, an ion pair reagent for cation analysis is used.
Examples of ion pair reagents for cation analysis include sulfonates or sulfates having an alkyl group having 3 to 20 carbon atoms. Specific examples include sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate, sodium dodecane sulfonate, sodium dodecyl sulfate, and the like.
[0012]
The conditions of high performance liquid chromatography using a reverse phase column in the second step are as follows.
The concentration of the ion pair reagent in the mobile phase is usually about 5 to 20 mM. The mixing ratio of the organic solvent (that is, the mixing ratio of the organic solvent with respect to the entire mobile phase) is 0 to 100%, and the mixing ratio is appropriately set according to the compound to be separated. As the reverse phase column, a reverse phase column in which a stationary phase in which octadecyl group, octyl group, etc. are chemically bonded to silica is packed is used, and the inner diameter of the column is usually about 2 to 5 mm ID. The thickness is usually about 50 to 250 mm. The flow rate of the mobile phase is usually about 0.1 to 2 mL / min. The injection amount may be set as appropriate according to the column diameter.
[0013]
In the third step of the method of the present invention, the secondary analysis sample separated in the second step is mixed with argon gas mixed with oxygen at a ratio of 1% (v / v) to 20% (v / v) as a carrier gas. And mass spectrometry (ICP-MS) using ionization means by inductively coupled plasma method may be used.
[0014]
In the mass spectrometry (ICP-MS) using ionization means by the inductively coupled plasma method in the third step, examples of a method for ionizing elements include a method for generating argon plasma.
[0015]
The conditions of mass spectrometry (ICP-MS) using ionization means by the above inductively coupled plasma method are as follows.
Argon gas mixed with oxygen in a ratio of 1% (v / v) to 20% (v / v), preferably 3% (v / v) to 15% (v / v) is used as a carrier gas.
The carrier gas is prepared by mixing argon gas and oxygen gas before introduction into the ICP-MS apparatus.
[0016]
By subjecting the platinum-containing compound contained in the secondary analysis sample to a mass spectrometry method (ICP-MS) using ionization means by inductively coupled plasma method, the secondary analysis sample is derived from platinum element m / z = 194,195,196 The amount of platinum is quantified by detecting and comparing the integrated value of the obtained peak with a calibration curve.
[0017]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited to an Example.
Analysis conditions <HPLC conditions>
Mobile phase: Gradient of 1 mM sodium heptanesulfonate aqueous solution and methanol (1) Gradient condition A (used in Example 1)
0 min. 100/0, 10 min. 100/0, 20 min. 0/100, 35 min. 0/100,
36 min. 100/0, 50 min. 100/0 (1 mM sodium heptanesulfonate aqueous solution / methanol)
(2) Gradient condition B (used in Example 2);
0 min. 100/0, 10 min. 100/0, 20 min. 80/20, 25 min. 75/25,
38 / min. 70/30, 40 min. 56/44, 48 min. 56/44, 62 min. 50/50, 64 min. 0/100, 90 min. 0/100 (1 mM sodium heptanesulfonate aqueous solution / methanol)
Detection: ICP-MS (m / z = 195 (Pt))
Column: ODS column <ICP-MS conditions>
ICP-MS: Agilent 4500 (Agilent)
Plasma output: 1.56 to 1.60 kW
Sampling position: 7.5 to 8.5 mm
Argon gas flow Plasma gas: About 16 L / min
Auxiliary gas: About 1.0 L / min
Carrier gas: approx. 0.9 to 1.1 L / min
Oxygen gas introduction amount in carrier gas: 5% (V / V) to 10% (V / V)
Peristaltic pump rotation speed: 0.1 rps (use only on drain side)
Spray chamber (S / C) temperature: 1-2 ℃
Normal introduction nebulizer (Sample Uptake approx. 1.2 mL / min)
Stabilization time from plasma lighting to measurement start: 40 minutes or more [0018]
Example 1 (Example of analysis of compound (A) contained in a biological sample: calculation of lower limit of detection and lower limit of quantification)
Serum from rats not receiving platinum-containing compounds (ie, blank rats) (Crj: CD (SD) male rat 7-week-old pooled serum (Oriental Yeast Co., Ltd.) was sent to Centrifree YM-30 (Amicon) ) And centrifuged at 4 ° C. and 2000 × g for 20 minutes (HIMAC CR20B3, manufactured by Hitachi, Ltd.) to obtain an ultrafiltrate.
Compound (A) was added to the obtained ultrafiltrate so as to have concentrations of 0, 0.5, 1, 5, 10, 30, and 50 ng / mL (indicated by the concentration of compound (A)). By subjecting this (100 μl) to high performance liquid chromatography in accordance with the above HPLC conditions, the secondary analysis sample is separated, and the separated secondary analysis sample is further subjected to inductively coupled plasma method in accordance with the above ICP-MS conditions. By subjecting it to mass spectrometry using an ionization means, the platinum-containing compound contained in the secondary analysis sample was detected element-specifically as a platinum element. The obtained charts are shown in FIGS.
A calibration curve was created using the integrated value of the peak corresponding to the compound (A) in the obtained chart. The results are shown in FIG. As is clear from FIG. 1, linearity was confirmed in the concentration range of 0.5 ng / mL to 50 ng / mL. The lower limit of quantification was 0.025 ng of Pt, and the lower limit of detection calculated from noise was 0.0025 ng of Pt. The lower limit of detection and the lower limit of quantification were determined as follows.
<Calculation method of lower limit of quantification and lower limit of detection>
Lower detection limit: The lower detection limit is defined as the peak signal (S) that has a significant difference from the baseline noise (N), and a signal (S = 3N) that has three times the noise intensity is used as the lower detection limit. Calculated as follows.
In Figure 2 (0.5 ng eq. Of compound (A) / mL, sample volume 100 μL, 0.025 ng eq. Of Pt), the baseline noise is about 4 abundance and the signal intensity of compound (A) is about 108 abundance. Therefore, the lower limit of detection is 4 × 3 × 0.5 (ng eq. Of compound (A) / mL) /108=0.05 ng eq. Of compound (A) / mL (sample amount 100 μL, 0.0025 ng eq. Of Pt).
When 0.05 eq. Of Compound (A) / mL was used for the measurement, the amount of Compound (A) in the total amount of the solution introduced into the detector was 0.05 ng eq. Of Compound (A) /mL×0.1 mL. = 0.005 ng eq. Of compound (A), and converted to the amount of platinum element, 0.005 ng eq. Of compound (A) = 0.005 × 195/380 = 0.0025 ng eq. Of Pt. Therefore, the lower limit of detection is 0.0025 ng in terms of the amount of platinum element.
Lower limit of quantification: The lower limit of quantification in FIG. 2 is 0.5 ng / mL (sample amount 100 μL) as the concentration of compound (A). The amount of the compound (A) in the total amount of the solution introduced into the detector at this time is 0.5 ng eq. Of the compound (A) /mL×0.1 mL = 0.05 ng eq. Of the compound (A). In terms of conversion, 0.05 ng eq. Of compound (A) = 0.05 × 195/380 = 0.025 ng eq. Of Pt. Therefore, the lower limit of quantification is 0.025 ng as the amount of platinum element.
[0019]
Example 2 (Analysis example of compound (B) contained in biological sample)
Lipiodol was added and suspended in a freeze-dried preparation of Compound (B) (manufactured by Sumitomo Pharmaceutical Co., Ltd.) to a concentration of 20 mg / mL. After adding 4 mL of rat serum, dog serum or human serum to 0.4 mL of the obtained suspension, the obtained mixture was shaken at 37 ° C., 5 rpm for 5 days. Five days later, lipiodol and serum were separated by centrifugation (HIMAC CR5DL and HIMAC CF8DL, manufactured by Hitachi, Ltd.) at 4 ° C. and 3000 rpm for 10 minutes. The separated serum is immediately collected in Centrifree YM-30 (Amicon) and centrifuged at 4 ° C and 2000 xg for 20 minutes (HIMAC CR20B3, Hitachi, Ltd.) to obtain an ultrafiltrate. Got. 100 μL of the obtained ultrafiltrate was used as a primary analysis sample, and the platinum-containing compound contained in the biological sample was analyzed with high sensitivity under the same conditions and method as in Example 1.
Rat serum is Crj: CD (SD) male rat 7 week old pool serum (Oriental Yeast Industry), dog serum is male Beagle dog serum (Oriental Yeast Industry), and human serum is quality control. A freeze-dried pool serum registered trademark Consera (manufactured by Nissui Pharmaceutical Co., Ltd.) was used.
As a result of the analysis, a peak corresponding to the methionine coordination compound of compound (A) (that is, a platinum-containing compound in which methionine is coordinated to platinum element) was detected, and the detection profile is shown in FIG.
[0020]
Example 3 (Analysis example of compound (B) contained in biological sample)
Lipiodol was added to a freeze-dried preparation of compound (B) (manufactured by Sumitomo Pharmaceutical Co., Ltd.) to a concentration of 2.5 mg / mL and suspended. The resulting suspension was subcutaneously administered to rats (Crj: CD (SD) male male rats 7 weeks old; (manufactured by Japan Charles River Co., Ltd.)) at a single subcutaneous dose of 50 mg / kg.
At 2, 6 and 24 hours after administration, the rats were anesthetized with ether and whole blood was collected from the posterior vena cava of the rats. Urine was collected for up to 24 hours after administration. The blood was allowed to stand for about 30 minutes under ice cooling, and then centrifuged at 4 ° C. and 3000 rpm for 10 minutes (HIMAC CR5DL and HIMAC CF8DL, manufactured by Hitachi, Ltd.) to obtain serum. The obtained serum was immediately collected in Centrifree YM-30 (Amicon), and then ultrafiltered by centrifugation (HIMAC CR20B3, Hitachi, Ltd.) for 20 minutes at 4 ° C and 2000 xg. A liquid was obtained. Using the obtained ultrafiltrate and 200 uL of urine as a primary analysis sample, the platinum-containing compound contained in the biological sample was analyzed with high sensitivity under the same conditions and method as in Example 1 except for the following conditions.
As a result of the analysis, a peak corresponding to the methionine coordination compound of compound (A) (that is, a platinum-containing compound in which methionine is coordinated to platinum element) was detected, and the detection profiles are shown in FIGS.
<Analysis conditions>
Mobile phase: 1 mM sodium heptanesulfonate aqueous solution and methanol gradient (0/100 (v / v) to 100/0 (v / v))
Column: ODS column <ICP-MS conditions>
ICP-MS: Agilent 4500 (Agilent)
Plasma output: 1.2 to 1.6kW
Sampling position: 0.75 to 12.0mm
Oxygen gas introduction amount in carrier gas: 5% (V / V) to 10% (V / V)
Carrier gas flow rate: 0.75 to 1.10L / min.
The lower limit of quantification was 0.025 ng of Pt.
[0021]
【The invention's effect】
According to the present invention, a platinum-containing compound contained in a biological sample can be analyzed with ultra-high sensitivity without requiring special equipment and equipment for handling a radiolabeled compound, and without requiring an advanced operation by an expert. became.
[Brief description of the drawings]
FIG. 1 (a) Calibration curve for addition of compound (A) (0.5 to 10 ng / mL).
(b) Calibration curve with addition of compound (A) (0.5-50 ng / mL).
FIG. 2 is a standard curve for addition of compound (A) (0.5 to 10 ng / mL).
FIG. 3 is a calibration curve with addition of compound (A) (10 to 50 ng / mL).
FIG. 4 shows an LC-ICP-MS measurement result (rat) of an in vitro reaction product of compound (B).
FIG. 5 shows LC-ICP-MS measurement results of rat serum ultrafiltrate after administration of compound (B) (2 hours after administration).
FIG. 6 shows LC-ICP-MS measurement results of rats after administration of compound (B) (24 hours after administration).

Claims (6)

A method for analyzing a platinum-containing compound contained in a biological sample,
(1) By subjecting a biological sample containing a platinum-containing compound to pretreatment for use in high performance liquid chromatography (HPLC) using a reverse phase column, or by directly using a biological sample containing a platinum-containing compound, A first step of preparing an analytical sample,
(2) The primary analysis sample prepared in the first step is a reversed-phase column in which the difference in solubility of the platinum-containing compound from the stationary phase to the mobile phase is controlled, and the organic solvent-water ion pair reagent added mobile phase is A second step of separating the secondary analysis sample by subjecting it to high performance liquid chromatography using a reverse phase column in which the elution power of is controlled by increasing the mixing ratio of the organic solvent in a gradient manner,
(3) The secondary analysis sample separated in the second step uses an argon gas mixed with oxygen in a ratio of 1% (v / v) to 20% (v / v) as a carrier gas, and is inductively coupled. A third step of element-specific detection of platinum-containing compounds contained in the secondary analysis sample as platinum elements by subjecting to mass spectrometry (ICP-MS) using ionization means by plasma method,
A method characterized by comprising: The platinum-containing compound contained in the biological sample is a [(1R, 2R) -1,2-cyclohexanediamine-N, N '] platinum compound or a platinum-containing metabolite of the compound in vivo. The method according to claim 1. 3. The method according to claim 2, wherein the platinum-containing metabolite in the living body is a platinum-containing compound obtained by coordinating amino acid to platinum element. The method according to claim 1 or 2, wherein the biological sample is serum, plasma, urine, or tissue collected from a mammal. The method according to claim 1, 2, 3, or 4, wherein the ion pair reagent used in the second step is an ion pair reagent for cation analysis. 6. The method according to claim 5, wherein the ion pair reagent for cation analysis is a sulfonate or sulfate having an alkyl group having 3 to 20 carbon atoms.

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