JP3479655B2 - Method for measuring bilirubin by high performance liquid chromatography - Google Patents
Method for measuring bilirubin by high performance liquid chromatographyInfo
- Publication number
- JP3479655B2 JP3479655B2 JP25833395A JP25833395A JP3479655B2 JP 3479655 B2 JP3479655 B2 JP 3479655B2 JP 25833395 A JP25833395 A JP 25833395A JP 25833395 A JP25833395 A JP 25833395A JP 3479655 B2 JP3479655 B2 JP 3479655B2
- Authority
- JP
- Japan
- Prior art keywords
- bilirubin
- solution
- internal standard
- type
- present
- 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.)
- Expired - Lifetime
Links
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 title claims description 100
- 238000000034 method Methods 0.000 title claims description 24
- 238000004128 high performance liquid chromatography Methods 0.000 title claims description 15
- 239000000126 substance Substances 0.000 claims description 13
- -1 flavin compound Chemical class 0.000 claims description 6
- 239000002798 polar solvent Substances 0.000 claims description 5
- 230000002378 acidificating effect Effects 0.000 claims description 4
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 17
- 238000000926 separation method Methods 0.000 description 8
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 7
- 239000011768 flavin mononucleotide Substances 0.000 description 7
- 229940013640 flavin mononucleotide Drugs 0.000 description 7
- FVTCRASFADXXNN-UHFFFAOYSA-N flavin mononucleotide Natural products OP(=O)(O)OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-UHFFFAOYSA-N 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 235000019231 riboflavin-5'-phosphate Nutrition 0.000 description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- BPYKTIZUTYGOLE-UHFFFAOYSA-N billirubin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(C=C3C(=C(C=C)C(=O)N3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-UHFFFAOYSA-N 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 5
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 description 5
- 239000011714 flavin adenine dinucleotide Substances 0.000 description 5
- 229940093632 flavin-adenine dinucleotide Drugs 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 2
- FPLYNRPOIZEADP-UHFFFAOYSA-N octylsilane Chemical compound CCCCCCCC[SiH3] FPLYNRPOIZEADP-UHFFFAOYSA-N 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 108010015428 Bilirubin oxidase Proteins 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 206010023129 Jaundice cholestatic Diseases 0.000 description 1
- 201000005267 Obstructive Jaundice Diseases 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 150000002211 flavins Chemical class 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 208000007475 hemolytic anemia Diseases 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000001052 yellow pigment Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Description
【発明の詳細な説明】
【０００１】
【発明の属する技術分野】本発明は、高速液体クロマト
グラフィ（以下、ＨＰＬＣともいう）によるビリルビン
の測定方法に関する。
【０００２】ビリルビンは、ヘモグロビンの代謝によっ
て生成し胆汁中に最も多く存在する黄色色素で、生体液
中では大別して非抱合型ビリルビン、及び抱合型ビリル
ビンとして存在する。ビリルビンの測定は各種疾病診断
の指標となる重要な検査対象項目で、臨床診断の分野に
おいては日常的にその測定が行われている。例えば、溶
血性貧血、溶血性黄疸などの病態では、主に非抱合型ビ
リルビン量が増大し、また閉塞性黄疸などの病態では抱
合型ビリルビン量が増大することが知られている。従っ
て、ビリルビンの測定は、臨床学的には総ビリルビンと
共に、前記個々（非抱合型ビリルビン、及び抱合型ビリ
ルビン）のビリルビンに分別して定量することが必要で
ある。
【０００３】
【従来の技術】ビリルビンの測定方法としては、ビリル
ビンとジアゾ試薬との反応により生成するアゾビリルビ
ンを比色定量する方法、ビリルビンオキシダーゼ等の酸
化酵素を用いた酵素的測定方法、ＨＰＬＣによる測定方
法等が知られている。即ち、総ビリルビンの測定方法と
しては特開昭５９−１７９９９号、特開昭６０−２４９
０６０号等、抱合型ビリルビンの測定方法としては特開
昭６０−１５２９５５号、特開昭６２−５８９９９号、
特開平２−２３８８９７号等、非抱合型ビリルビンの測
定方法としては特開昭６０−１５４１６２号等が開示さ
れている。
【０００４】
【発明が解決しようとする課題】ビリルビンの分別に関
して、前記の非抱合型ビリルビンと抱合型ビリルビンと
いう分別以外に、ＨＰＬＣにおいてはα型ビリルビン、
β型ビリルビン、γ型ビリルビン及びδ型ビリルビンと
いう分別がある。即ち、短時間に各ビリルビンを測定す
るに好適なＨＰＬＣ法は、ラウフ等（Ｊｏｕｒｎａｌ
ｏｆ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ、２２６，３９１
−４０２，１９８１）により確立されており、血清中の
ビリルビンが４分画に分けられることが報告されてい
る。この場合、α型ビリルビンは非抱合型ビリルビン
に、β型ビリルビンとγ型ビリルビンは抱合型ビリルビ
ンであり、δ型ビリルビンはどちらにも属さない。前述
のように、病態により各ビリルビン量を正確に把握する
ことが必要であり、ＨＰＬＣ法によるビリルビンの測定
はジアゾ法や酵素法とともに重要な手段として利用され
ている。
【０００５】このＨＰＬＣ法は、各ビリルビンのピーク
面積（あるいはピーク高さ）から測定値を計算する方法
のため、試料の注入量差、移動相組成の僅かな変化によ
る溶出時間の変動等が、測定誤差発生の大きな要因とし
て問題となっていた。本願発明者等は、ＨＰＬＣ法によ
ってビリルビンを測定する際に好適な内部標準物質を探
索・決定したところ、フラビン化合物を使用すれば、各
ビリルビン値を正確に測定することができることを知り
本発明を完成した。
【０００６】
【課題を解決するための手段】本願発明は以上の見地か
らなされたもので、次の請求項１から構成されている。
請求項１：酸性の極性溶媒を移動相とした逆相クロマト
グラフィによりビリルビンを検出する方法において、内
部標準物質としてフラビン化合物を用いることを特徴と
する高速液体クロマトグラフィによるビリルビンの測定
方法。
【０００７】本発明のＨＰＬＣ法の分析モードは、基本
的にラウフ等の方法に準ずるものである。好適な内部標
準物質としては、各ビリルビンのピークと重ならず十分
な分離度を有し、且つ測定系に干渉しないことが必要で
ある。そこで、種々の化合物について検討した結果、最
も好適な内部標準物質は、フラビン化合物であることを
見出した。
【発明の実施の形態】
【０００８】フラビン化合物としては、具体的にはリボ
フラビン、フラビンモノヌクレオチド（ＦＭＮ）、フラ
ビンアデニンジヌクレオチド（ＦＡＤ）である。例え
ば、フラビンモノヌクレオチドを内部標準物質とした場
合、α型ビリルビン、β型ビリルビン、γ型ビリルビン
及びδ型ビリルビンとの分離度は、それぞれ１０．２、
６．１、３．６、３．０（分離度ＲS = ２（ＶR1−
ＶR2) ／Ｗ1 + Ｗ2 として求めた。但し、ＶR は溶質の
保持容量、Ｗはピーク幅である。１，２は溶質を示し、
２はフラビンモノヌクレオチド）となり、また、反応系
への干渉も皆無であった。その添加量は、各ビリルビン
との面積比（あるいは高さ比）を考慮して決定すれば良
く、特に限定する必要はない。例えば、血清１容量に対
して、濃度０．１μＭ〜１ｍＭのフラビンモノヌクレオ
チドの水溶液を１容量添加する。
【０００９】本発明におけるＨＰＬＣの分析条件は、α
型ビリルビン、β型ビリルビン、γ型ビリルビン及びδ
型ビリルビンが分離される範囲内で、ラウフ等の分離モ
ードを変更することは可能である。ラウフ等の規定した
分析条件は以下の通りである。
カラム：リクロゾルブＲＰ−８（メルク社）、内径４ｍｍ×長さ２５０ｍｍ
溶離液Ａ：２−メトキシエタノールとｐＨ２．０の５０ｍＭリン酸緩衝液の混
液（５：９５）
溶離液Ｂ：イソプロピルアルコールと２−メトキシエタノールの混液（９５：
５）１０００ｍｌにリン酸２５ｍｌを添加した液。
条件：Ａ液→Ｂ液８０％リニアグラジェント（１６分）
Ｂ液８０％（８分）
Ｂ液８０％→Ａ液リニアグラジェント（７分）
温度：４１℃
流速：１．４ｍｌ／ｍｉｎ
検出：４５０ｎｍ
【００１０】例えば、カラムは上記リクロゾルブＲＰ−
８以外に、シリカゲルをオクチルシランで化学処理した
もの、オクタデシルシランで化学処理したものが使用で
きる。具体的には、シリカゲルをオクチルシランで化学
処理したものとして、ヌクレオシル−Ｃ8 （マチェリー
・ナーゲル社）、イナートシルＣ8 （ガスクロ工業）、
ショーデックス−Ｃ8 （昭和電工）等を挙げることがで
きる。また、シリカゲルをオクタデシルシランで化学処
理したものとして、リクロゾルブＲＰ−１８（メル
ク）、ＴＳＫ ＧＥＬ ＯＤＳ−１２０Ｔ（東ソー）、
ヌクレオシル−Ｃ18（マチェリー・ナーゲル社）、イナ
ートシルＣ18（ガスクロ工業）、ショーデックス−Ｃ18
（昭和電工）、ボンダパックＯＤＳ（ウォーターズ）、
日立ゲル＃３０５０（日立製作所）等を用いることがで
きる。
【００１１】例えば、溶離液は基本的に酸性の極性溶媒
を用いればよく、酸性物質としてはリン酸以外にクエン
酸、酒石酸等を、移動相の全組成中に１〜１０００ｍ
Ｍ、好ましくは１０〜５００ｍＭの濃度範囲内で適宜選
択すればよい。また、ｐＨは１．５〜６．５、好ましく
は２．０〜４．０となるように調整する。また、極性溶
媒としては、上記以外にメタノール、アセトニトリル、
エタノール、ジオキサン等を単独あるいは組み合わせて
用いることもできる。Ａ液及びＢ液における極性溶媒の
含有量は１〜９９％の範囲内で、各ビリルビンと内部標
準物質との分離度と溶出時間を考慮したうえで適宜選択
できる。
【００１２】本発明における試料は、血清、血漿、全
血、尿、唾液、髄液等の生体由来液であり、これを必要
に応じて前処理（例えば、除タンパク処理やプレカラム
処理等）し試料とする。例えば、血清を試料とする場合
には、特に前処理を施すことなく、本発明による内部標
準物質の溶液を一定量添加したものを直接あるいは濾過
した後注入することができる。あるいは、必要に応じて
更に溶離液等で希釈したものを注入すればよい。
【００１３】上記条件により得られた標準物質と試料の
各クロマトグラムの各フラクションピーク面積を半値幅
法、またはオートアナライザーによる自動計算によって
求め、各ビリルビンの面積を標準物質の面積で除した値
の比から、各ビリルビンの定量値を算出することができ
る。図１に内部標準物質を用いない従来法のクロマトグ
ラムを、図２に内部標準物質としてＦＡＤを添加した本
発明のクロマトグラムを、また図３に内部標準物質とし
てＦＭＮを添加した本発明のクロマトグラムの例を示
す。
【００１４】
【実施例】以下、実施例によって本発明を具体的に説明
するが、これらは本発明の範囲を限定するものではな
い。
＜実施例１＞
内部標準物質の選別と分離度
ヒト血清２０μｌに、ＦＡＤの０．１ｍＭ水溶液２０μ
ｌを加え、その混液を試料として２０μｌをＨＰＬＣに
注入し分析（条件は以下の通り）した。また、ＦＡＤに
換えて、ＦＭＮの０．１ｍＭ水溶液２０μｌを加え、同
様に分析した。それぞれの内部標準物質について、α型
ビリルビン、β型ビリルビン、γ型ビリルビン及びδ型
ビリルビンとの分離度を上述した計算式により求めた。
結果を表１に示す。表の通り、内部標準物質のピークと
ビリルビンの各フラクションピークとの分離は良好であ
った。
（ＨＰＬＣ分析条件）
カラム：リクロゾルブＲＰ−１８（メルク社）、内径４ｍｍ×長さ２５０ｍｍ
溶離液Ａ：２−メトキシエタノールとｐＨ２．１の５０ｍＭリン酸緩衝液の混
液（５：９５）
溶離液Ｂ：イソプロピルアルコールと２−メトキシエタノールの混液（９５：
５）１０００ｍｌにリン酸２５ｍｌを添加した液。
条件：Ａ液→Ｂ液８０％リニアグラジェント（１６分）
Ｂ液８０％（８分）
Ｂ液８０％→Ａ液リニアグラジェント（７分）
温度：４１℃
流速：１．０ｍｌ／ｍｉｎ
検出：４５０ｎｍ
装置：ＬＣ−６Ａ（島津製作所）、８０５データステーション（ウォーターズ
社）
【００１５】
【００１６】＜実施例２＞
従来法との精度比較
前記実施例１と同様のＨＰＬＣ分析条件により、ヒト血
清を用いて内部標準物質無添加のものと、内部標準物質
としてＦＡＤを加えたものについて、繰り返し操作によ
る再現性試験を行った。なお、各ビリルビンフラクショ
ンの測定値（ｍｇ／ｄｌ）は、標準品としてジタウロビ
リルビン（ポルフィリンプロダクト社）の１０ｍｇ／ｍ
ｌ濃度の溶液を用いて同様の操作を行い求めた。結果を
表２に示す。結果の通り、内部標準を用いない従来法の
精度も満足いくものではあったが、本発明は更に精度が
向上した。
【００１７】
【００１８】
【発明の効果】本発明を利用することにより、ＨＰＬＣ
法による各ビリルビンの定量精度が大幅に向上し、疾病
診断の指標として有用なデータを供することができると
いう効果を有する。Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring bilirubin by high performance liquid chromatography (hereinafter, also referred to as HPLC). [0002] Bilirubin is a yellow pigment produced by the metabolism of hemoglobin and most frequently present in bile, and is roughly classified in biological fluids as unconjugated bilirubin and conjugated bilirubin. The measurement of bilirubin is an important test item that serves as an index for diagnosis of various diseases, and the measurement is routinely performed in the field of clinical diagnosis. For example, it is known that in pathological conditions such as hemolytic anemia and hemolytic jaundice, the amount of unconjugated bilirubin mainly increases, and in pathological conditions such as obstructive jaundice, the amount of conjugated bilirubin increases. Therefore, the measurement of bilirubin is clinically required to be quantified separately from the total (unconjugated bilirubin and conjugated bilirubin) together with the total bilirubin. [0003] As a method for measuring bilirubin, a method for colorimetric determination of azobilirubin produced by the reaction of bilirubin with a diazo reagent, a method for enzymatic measurement using an oxidase such as bilirubin oxidase, and a method for HPLC. Measurement methods and the like are known. That is, methods for measuring total bilirubin are described in JP-A-59-17999 and JP-A-60-249.
060 and the like, as methods for measuring conjugated bilirubin, include JP-A-60-152950, JP-A-62-58999,
JP-A-2-238897 discloses a method for measuring unconjugated bilirubin, and JP-A-60-154162 discloses the method. [0004] Regarding the separation of bilirubin, in addition to the above-mentioned separation of unconjugated bilirubin and conjugated bilirubin, α-type bilirubin,
There is a classification as β-type bilirubin, γ-type bilirubin, and δ-type bilirubin. That is, an HPLC method suitable for measuring each bilirubin in a short time is described in Rauff et al.
of Chromatography, 226,391
-402, 1981), and reports that bilirubin in serum is divided into four fractions. In this case, α-type bilirubin is unconjugated bilirubin, β-type and γ-type bilirubin are conjugated bilirubins, and δ-type bilirubin does not belong to either. As described above, it is necessary to accurately determine the amount of each bilirubin depending on the disease state, and the measurement of bilirubin by the HPLC method is used as an important means together with the diazo method and the enzyme method. In this HPLC method, the measured value is calculated from the peak area (or peak height) of each bilirubin. This has been a problem as a major factor in the occurrence of measurement errors. The present inventors have searched and determined a suitable internal standard when measuring bilirubin by the HPLC method, and found that if a flavin compound is used, each bilirubin value can be accurately measured, and the present invention was found. completed. SUMMARY OF THE INVENTION The present invention has been made in view of the above, and is constituted by the following claim 1. Claim 1: A method for detecting bilirubin by reverse phase chromatography using an acidic polar solvent as a mobile phase, wherein a flavin compound is used as an internal standard substance. [0007] The analysis mode of the HPLC method of the present invention basically conforms to the method of Rauch et al. As a suitable internal standard substance, it is necessary that it does not overlap with the peak of each bilirubin, has sufficient resolution, and does not interfere with the measurement system. Then, as a result of examining various compounds, it was found that the most suitable internal standard substance was a flavin compound. DETAILED DESCRIPTION OF THE INVENTION The flavin compounds are specifically riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). For example, when a flavin mononucleotide is used as an internal standard, the degree of separation from α-type bilirubin, β-type bilirubin, γ-type bilirubin and δ-type bilirubin is 10.2, respectively.
6.1,3.6,3.0 (separation degree R S = 2 (V R1 -
V R2 ) / W 1 + W 2 . However, V R is the retention volume of solute, W is the peak width. 1 and 2 indicate solutes,
2 was a flavin mononucleotide), and there was no interference with the reaction system. The addition amount may be determined in consideration of the area ratio (or height ratio) with each bilirubin, and need not be particularly limited. For example, 1 volume of an aqueous solution of flavin mononucleotide having a concentration of 0.1 μM to 1 mM is added to 1 volume of serum. In the present invention, the HPLC analysis condition is α
-Type bilirubin, β-type bilirubin, γ-type bilirubin and δ
It is possible to change the separation mode, such as Rauff, within the range in which type bilirubin is separated. The analytical conditions specified by Rauff et al. Are as follows. Column: Lichrosolv RP-8 (Merck), inner diameter 4 mm x length 250 mm Eluent A: mixture of 2-methoxyethanol and 50 mM phosphate buffer at pH 2.0 (5:95) Eluent B: isopropyl alcohol A solution obtained by adding 25 ml of phosphoric acid to 1000 ml of a mixed solution (95: 5) of 2-methoxyethanol. Condition: Solution A → Solution B 80% linear gradient (16 minutes) Solution B 80% (8 minutes) Solution B 80% → Solution A linear gradient (7 minutes) Temperature: 41 ° C Flow rate: 1.4 ml / min Detection The column is, for example, Lichrosolve RP-
In addition to 8, those obtained by chemically treating silica gel with octylsilane and those chemically treated with octadecylsilane can be used. Specifically, as silica gel chemically treated with octylsilane, Nucleosyl-C8 (Machery Nagel), Inertosyl C8 (Gascro Industry),
Shodex-C8 (Showa Denko) and the like. Further, assuming that silica gel is chemically treated with octadecylsilane, Lichrosolve RP-18 (Merck), TSK GEL ODS-120T (Tosoh),
Nucleosil-C18 (Machery Nagel), Inertosyl C18 (Gascro Industry), Shodex-C18
(Showa Denko), Bonder Pack ODS (Waters),
Hitachi Gel # 3050 (Hitachi, Ltd.) or the like can be used. For example, an acidic polar solvent may be basically used as the eluent, and citric acid, tartaric acid, etc., other than phosphoric acid, may be used as an acidic substance in the total mobile phase composition in an amount of 1 to 1000 m.
M, preferably within a concentration range of 10 to 500 mM. Further, the pH is adjusted to be 1.5 to 6.5, preferably 2.0 to 4.0. In addition, as the polar solvent, other than the above, methanol, acetonitrile,
Ethanol, dioxane and the like can be used alone or in combination. The content of the polar solvent in the solution A and the solution B can be appropriately selected in the range of 1 to 99% in consideration of the degree of separation between each bilirubin and the internal standard and the elution time. The sample in the present invention is a biologically derived fluid such as serum, plasma, whole blood, urine, saliva, cerebrospinal fluid, etc., and is subjected to pretreatment (for example, protein removal treatment or precolumn treatment) as necessary. Use as a sample. For example, when serum is used as a sample, a sample to which a fixed amount of the internal standard substance solution according to the present invention has been added can be injected directly or after filtration without any particular pretreatment. Alternatively, if necessary, a solution diluted with an eluent or the like may be injected. [0013] The area of each fraction peak in each chromatogram of the standard substance and the sample obtained under the above conditions is determined by a half width method or automatic calculation by an autoanalyzer, and the area of each bilirubin divided by the area of the standard substance is calculated. From the ratio, the quantitative value of each bilirubin can be calculated. FIG. 1 shows a chromatogram of a conventional method without using an internal standard, FIG. 2 shows a chromatogram of the present invention to which FAD was added as an internal standard, and FIG. 3 shows a chromatogram of the present invention to which FMN was added as an internal standard. Here is an example of a gram. The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. <Example 1> Selection and resolution of internal standard substance 20 μl of human serum was added to 20 μl of 0.1 mM aqueous solution of FAD.
1 was added, and the mixture was used as a sample, and 20 μl of the mixture was injected into HPLC and analyzed (conditions are as follows). In addition, instead of FAD, 20 μl of a 0.1 mM aqueous solution of FMN was added, and analysis was performed in the same manner. For each of the internal standard substances, the degree of separation from α-type bilirubin, β-type bilirubin, γ-type bilirubin, and δ-type bilirubin was determined by the above formula.
Table 1 shows the results. As shown in the table, the separation between the peak of the internal standard substance and each fraction peak of bilirubin was good. (HPLC analysis conditions) Column: Lichrosolv RP-18 (Merck), inner diameter 4 mm x length 250 mm Eluent A: mixture of 2-methoxyethanol and 50 mM phosphate buffer of pH 2.1 (5:95) Eluent B: A solution obtained by adding 25 ml of phosphoric acid to 1000 ml of a mixed solution (95: 5) of isopropyl alcohol and 2-methoxyethanol. Conditions: Solution A → Solution B 80% linear gradient (16 minutes) Solution B 80% (8 minutes) Solution B 80% → Solution A linear gradient (7 minutes) Temperature: 41 ° C Flow rate: 1.0 ml / min Detection : 450 nm Apparatus: LC-6A (Shimadzu), 805 Data Station (Waters) <Example 2> Comparison of Accuracy with Conventional Method Under the same HPLC analysis conditions as in Example 1 above, a human serum was used without addition of an internal standard substance, and a FAM was added as an internal standard substance. And a reproducibility test by a repetitive operation was performed. The measured value (mg / dl) of each bilirubin fraction was 10 mg / m2 of ditaurobilirubin (porphyrin product) as a standard product.
The same operation was performed using a 1-concentration solution. Table 2 shows the results. As shown in the results, the accuracy of the conventional method using no internal standard was satisfactory, but the accuracy of the present invention was further improved. [0017] EFFECT OF THE INVENTION By utilizing the present invention, HPLC
This method has the effect of significantly improving the accuracy of quantification of each bilirubin by the method and providing useful data as an index for disease diagnosis.
【図面の簡単な説明】 【図１】従来法のクロマトグラムを示す図である。 【図２】本発明のクロマトグラムを示す図である。 【図３】本発明のクロマトグラムを示す図である。[Brief description of the drawings] FIG. 1 is a diagram showing a chromatogram according to a conventional method. FIG. 2 is a diagram showing a chromatogram of the present invention. FIG. 3 is a diagram showing a chromatogram of the present invention.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭59−203954（ＪＰ，Ａ) 特開 昭63−163275（ＪＰ，Ａ) 特開 昭62−58166（ＪＰ，Ａ) 特開 平３−99259（ＪＰ，Ａ) (58)調査した分野(Int.Cl.7，ＤＢ名) G01N 30/00 - 30/96 ──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-59-203954 (JP, A) JP-A-63-163275 (JP, A) JP-A-62-58166 (JP, A) 99259 (JP, A) (58) Field surveyed (Int. Cl. 7 , DB name) G01N 30/00-30/96
Claims (1)
マトグラフィによりビリルビンを検出する方法におい
て、内部標準物質としてフラビン化合物を用いることを
特徴とする高速液体クロマトグラフィによるビリルビン
の測定方法。(1) A method for detecting bilirubin by reverse phase chromatography using an acidic polar solvent as a mobile phase, wherein a flavin compound is used as an internal standard substance. Method for measuring bilirubin by HPLC.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25833395A JP3479655B2 (en) | 1995-09-12 | 1995-09-12 | Method for measuring bilirubin by high performance liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25833395A JP3479655B2 (en) | 1995-09-12 | 1995-09-12 | Method for measuring bilirubin by high performance liquid chromatography |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0980041A JPH0980041A (en) | 1997-03-28 |
| JP3479655B2 true JP3479655B2 (en) | 2003-12-15 |
Family
ID=17318793
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP25833395A Expired - Lifetime JP3479655B2 (en) | 1995-09-12 | 1995-09-12 | Method for measuring bilirubin by high performance liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3479655B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108918712A (en) * | 2018-07-18 | 2018-11-30 | 安徽科宝生物工程有限公司 | A kind of detection method of content of bilirubin |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114527218A (en) * | 2022-02-24 | 2022-05-24 | 北京五和博澳药业股份有限公司 | Method for detecting components of scutellaria baicalensis |
-
1995
- 1995-09-12 JP JP25833395A patent/JP3479655B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108918712A (en) * | 2018-07-18 | 2018-11-30 | 安徽科宝生物工程有限公司 | A kind of detection method of content of bilirubin |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0980041A (en) | 1997-03-28 |
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