JP6255234B2 - Method for measuring biotin or related substances in biological samples - Google Patents
Method for measuring biotin or related substances in biological samples Download PDFInfo
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- JP6255234B2 JP6255234B2 JP2013262185A JP2013262185A JP6255234B2 JP 6255234 B2 JP6255234 B2 JP 6255234B2 JP 2013262185 A JP2013262185 A JP 2013262185A JP 2013262185 A JP2013262185 A JP 2013262185A JP 6255234 B2 JP6255234 B2 JP 6255234B2
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 title claims description 239
- 229960002685 biotin Drugs 0.000 title claims description 118
- 235000020958 biotin Nutrition 0.000 title claims description 118
- 239000011616 biotin Substances 0.000 title claims description 118
- 238000000034 method Methods 0.000 title claims description 78
- 239000012472 biological sample Substances 0.000 title claims description 41
- 239000000126 substance Substances 0.000 title claims description 41
- 239000000523 sample Substances 0.000 claims description 132
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 129
- 239000007788 liquid Substances 0.000 claims description 67
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- 238000000502 dialysis Methods 0.000 claims description 56
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- 238000001631 haemodialysis Methods 0.000 claims description 32
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- 238000005259 measurement Methods 0.000 claims description 19
- 230000003993 interaction Effects 0.000 claims description 18
- 102000004169 proteins and genes Human genes 0.000 claims description 17
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- 125000000524 functional group Chemical group 0.000 claims description 8
- 208000001647 Renal Insufficiency Diseases 0.000 claims description 6
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- 201000001474 proteinuria Diseases 0.000 claims description 3
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 12
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- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 8
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 6
- 235000019253 formic acid Nutrition 0.000 description 6
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
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- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- NZERRTYPTPRCIR-ZKWXMUAHSA-N Alpha-Dehydrobiotin Natural products N1C(=O)N[C@@H]2[C@H](CCC=CC(=O)O)SC[C@@H]21 NZERRTYPTPRCIR-ZKWXMUAHSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
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- QDFGCLSCEPNVQP-VPLCAKHXSA-N Bisnorbiotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCC(=O)O)SC[C@@H]21 QDFGCLSCEPNVQP-VPLCAKHXSA-N 0.000 description 1
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- 102000004190 Enzymes Human genes 0.000 description 1
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- ITHUGYGPPSVHRH-FXQIFTODSA-N Methyl bisnorbiotinyl ketone Chemical compound N1C(=O)N[C@@H]2[C@H](CCC(=O)C)SC[C@@H]21 ITHUGYGPPSVHRH-FXQIFTODSA-N 0.000 description 1
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Description
本発明は、生体試料中のビオチンまたはその関連物質の測定方法、より詳細には、高速液体クロマトグラフィー（ＨＰＬＣ）を利用した生体試料（例えば、血漿、透析廃液、尿）中のビオチンまたはその関連物質の測定方法に関する。 The present invention relates to a method for measuring biotin or a related substance in a biological sample, and more particularly, biotin or a related substance in a biological sample (eg, plasma, dialysis waste liquid, urine) using high performance liquid chromatography (HPLC). It relates to a method for measuring substances.
ビオチンは、ビタミンＢ群に属する水溶性ビタミンの一種であり、ビタミンＢ７またはビタミンＨとも呼ばれている。ビオチンは、生体内でカルボキシラーゼ反応の補酵素として、呼吸系、β-酸化、脂肪酸生合成などにおいて機能することが知られている。 Biotin is a kind of water-soluble vitamin belonging to the vitamin B group and is also called vitamin B7 or vitamin H. Biotin is known to function in the respiratory system, β-oxidation, fatty acid biosynthesis and the like as a coenzyme for carboxylase reaction in vivo.
近年、ビオチンと種々の疾患との関係が明らかになりつつある。例えば、ビオチンは、アトピー性皮膚炎、掌蹠膿疱症等の皮膚疾患の治療に有効であること、血液透析による四肢の痙攣に対する抑制・予防効果があること、肝硬変患者における高アンモニア血症改善、耐糖能およびインスリン抵抗性の改善、血圧上昇抑制などの効果があることが知られている。他にも、先天的なビオチン代謝酵素欠損や抗痙攣薬の長期投与、特殊調製粉乳の摂取乳児等にビオチン欠乏症が発症することが知られている（特許文献１、非特許文献１〜１０など）。
したがって、健康管理や疾患の診断、ビオチンの体内動態に関する研究のため、血漿や透析廃液、尿などの生体試料中のビオチンおよびビオチン関連物質を迅速かつ簡便に高感度で測定することが必要とされている。
In recent years, the relationship between biotin and various diseases is becoming clear. For example, biotin is effective in the treatment of skin diseases such as atopic dermatitis and palmoplantar pustulosis, has an inhibitory / preventive effect on limb spasms caused by hemodialysis, improves hyperammonemia in patients with cirrhosis, It is known to have effects such as improvement of glucose tolerance and insulin resistance and suppression of blood pressure increase. In addition, it is known that biotin deficiency develops in congenital biotin-metabolizing enzyme deficiency, long-term administration of anticonvulsants, infants taking specially prepared milk powder, etc. (
Therefore, it is necessary to measure biotin and biotin-related substances in biological samples such as plasma, dialysis waste fluid, and urine quickly and easily with high sensitivity for research on health care, disease diagnosis, and biotin pharmacokinetics. ing.
一方、ビオチンの測定法としては、微生物学的定量法、結合アッセイ法、ＨＰＬＣ法、光学的測定法などが知られている。しかしながら、微生物学的定量法は精度が低い、操作が煩雑で時間を要することなどの問題があり、結合アッセイ法はビオチン関連物質（例えば、ビオチン代謝物）とも非特異的に反応するなどの問題があり、光学的測定法およびＨＰＬＣ法は感度が低いなどの問題がある（非特許文献１１）。
また、ＨＰＬＣ法で感度を高めるため、プレカラムやポストカラムによる誘導体化などが採用されているが（特許文献２など）、工程が煩雑であり、簡便ではなかった。
On the other hand, as a biotin measurement method, a microbiological quantification method, a binding assay method, an HPLC method, an optical measurement method, and the like are known. However, the microbiological quantification method has problems such as low accuracy, complicated operation, and time. The binding assay method also reacts non-specifically with biotin-related substances (for example, biotin metabolites). However, the optical measurement method and the HPLC method have problems such as low sensitivity (Non-patent Document 11).
In addition, derivatization with a pre-column or a post-column has been adopted in order to increase sensitivity in the HPLC method (
また、高速液体クロマトグラフ／質量分析法（ＬＣ／ＭＳ）または高速液体クロマトグラフ／タンデム質量分析法（ＬＣ／ＭＳ／ＭＳ）を用いたビオチンの測定法も知られている（非特許文献１２〜２３）。しかしながら、これらの文献に記載の測定法は、主として食品などを対象としたものであり、また、血漿や透析廃液、尿などの生体試料中のビオチンおよびビオチン関連物質を迅速かつ簡便に高感度で測定することができるものではなかった。 In addition, a biotin measurement method using high performance liquid chromatography / mass spectrometry (LC / MS) or high performance liquid chromatography / tandem mass spectrometry (LC / MS / MS) is also known (Non-Patent Documents 12- 23). However, the measurement methods described in these documents are mainly intended for foods and the like, and biotin and biotin-related substances in biological samples such as plasma, dialysis waste liquid, and urine are rapidly and easily and highly sensitive. It could not be measured.
本発明は、血漿や透析廃液、尿などの生体試料中のビオチンおよびビオチン関連物質を迅速かつ簡便に高感度で測定する方法を提供することを課題とするものである。 An object of the present invention is to provide a method for measuring biotin and biotin-related substances in biological samples such as plasma, dialysis waste liquid, and urine quickly and easily with high sensitivity.
本発明者らは、上記課題を解決するために鋭意検討した結果、生体試料中のタンパク質を除去した後、高速液体クロマトグラフ／タンデム質量分析法を用いて測定することで、生体試料中のビオチンおよびビオチン関連物質を迅速かつ簡便に高感度で測定することができることを見出し、本発明を完成させるに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have removed biotin in a biological sample and then measured it using high performance liquid chromatograph / tandem mass spectrometry, thereby obtaining biotin in the biological sample. Furthermore, the present inventors have found that biotin-related substances can be measured quickly and easily with high sensitivity, and the present invention has been completed.
したがって、本発明は、以下の態様を含む。
［１］ 生体試料中のビオチンまたはビオチン関連物質を測定する方法であって、
（１）生体試料中のタンパク質を除去する工程と、
（２）工程（１）で得られた試料中のビオチンまたはビオチン関連物質を、高速液体クロマトグラフ／タンデム質量分析法を用いて測定する工程と
を含む、方法。
［２］ 生体試料が血漿、透析廃液または尿である、上記［１］に記載の方法。
［３］ 生体試料が血漿である、上記［２］に記載の方法。
［４］ 生体試料が透析廃液である、上記［２］に記載の方法。
［５］ 生体試料が尿である、上記［２］に記載の方法。
［６］ 血漿が血液透析患者由来の血漿である、上記［３］に記載の方法。
［７］ 透析廃液が血液透析患者由来の透析廃液である、上記［４］に記載の方法。
［８］ 透析廃液が血液透析濾過患者由来の透析廃液である、上記［４］に記載の方法。
［９］ 透析廃液が腹膜透析患者由来の透析廃液である、上記［４］に記載の方法。
［１０］ 尿が腎不全患者由来の尿である、上記［５］に記載の方法。
［１１］ 尿がタンパク尿である、上記［５］に記載の方法。
［１２］ 工程（１）が、メタノールを用いてタンパク質を沈殿させることにより除去する工程である、上記［１］〜［１１］のいずれかに記載の方法。
［１３］ 高速液体クロマトグラフに使用するカラムが、疎水性相互作用、双極子-双極子相互作用、およびπ-π相互作用により分離するカラムである、上記［１］〜［１２］のいずれかに記載の方法。
［１４］ カラム担体の官能基が、ペンタフルオロフェニル基が結合した直鎖または分岐鎖のアルキル基を含む、上記［１２］に記載の方法。
［１５］ 生体試料中のビオチンまたはビオチン関連物質を測定するための測定用キットであって、
（１）生体試料中のタンパク質を除去する工程に使用するための試薬と、
（２）高速液体クロマトグラフ／タンデム質量分析法に使用するための機器
を含む、キット。
Accordingly, the present invention includes the following aspects.
[1] A method for measuring biotin or a biotin-related substance in a biological sample,
(1) removing a protein in the biological sample;
(2) measuring the biotin or biotin-related substance in the sample obtained in step (1) using high performance liquid chromatography / tandem mass spectrometry.
[2] The method according to [1] above, wherein the biological sample is plasma, dialysis waste liquid or urine.
[3] The method according to [2] above, wherein the biological sample is plasma.
[4] The method according to [2] above, wherein the biological sample is a dialysis waste liquid.
[5] The method according to [2] above, wherein the biological sample is urine.
[6] The method according to [3] above, wherein the plasma is plasma derived from a hemodialysis patient.
[7] The method according to [4] above, wherein the dialysis waste liquid is a dialysis waste liquid derived from a hemodialysis patient.
[8] The method according to [4] above, wherein the dialysis waste liquid is a dialysis waste liquid derived from a hemodiafiltration patient.
[9] The method according to [4] above, wherein the dialysis waste liquid is a dialysis waste liquid derived from a peritoneal dialysis patient.
[10] The method according to [5] above, wherein the urine is urine derived from a patient with renal failure.
[11] The method according to [5] above, wherein the urine is proteinuria.
[12] The method according to any one of [1] to [11] above, wherein the step (1) is a step of removing the protein by precipitation using methanol.
[13] Any of the above [1] to [12], wherein the column used for the high performance liquid chromatograph is a column that is separated by hydrophobic interaction, dipole-dipole interaction, and π-π interaction. The method described in 1.
[14] The method according to [12] above, wherein the functional group of the column carrier includes a linear or branched alkyl group to which a pentafluorophenyl group is bonded.
[15] A measurement kit for measuring biotin or a biotin-related substance in a biological sample,
(1) a reagent for use in the step of removing proteins in a biological sample;
(2) A kit comprising equipment for use in high performance liquid chromatograph / tandem mass spectrometry.
本発明の測定法によれば、生体試料中のビオチンおよびビオチン関連物質を迅速かつ簡便に高感度で測定することができる。
すなわち、本発明の測定法の感度は、市販されている代表的な結合アッセイキットと同等の感度でビオチンおよびビオチン関連物質を測定が可能である。また、ＨＰＬＣでの分離に加えて、ＭＲＭ法（Ｍｕｌｔｉｐｌｅ Ｒｅａｃｔｉｏｎ Ｍｏｎｉｔｏｒｉｎｇ：多重反応モニタリング）で分子量によるフィルタリングも行なっているので、微生物学的定量法や結合アッセイなどのように、ビオチンとビオチン関連物質（ビオチン代謝物など）との交差性はほとんどないと考えられる。
また、試料の前処理は、除タンパク質法であり、従来のＨＰＬＣによるビオチン血漿測定法の前処理において使用されていた誘導体化などが不要であるので、容易かつ短時間で前処理を行うことができる。
従来のＨＰＬＣ法では、ヒト血漿の場合、ビオチンの保持時間は早いものでも７〜８分であり、１検体の測定にほとんどが数十分から１時間程度の時間を要していたが、本発明の測定法では、１検体の分析サイクルは５分程度（ビオチンの保持時間は２分未満）と短いため、効率的な多検体処理が可能である。
According to the measurement method of the present invention, biotin and a biotin-related substance in a biological sample can be measured quickly and easily with high sensitivity.
That is, the sensitivity of the measurement method of the present invention can measure biotin and a biotin-related substance with the same sensitivity as a typical binding assay kit commercially available. In addition to HPLC separation, MRM (Multiple Reaction Monitoring) is also used to filter by molecular weight, so biotin and biotin-related substances (such as microbiological quantification and binding assays) It is considered that there is little cross-reactivity with biotin metabolites.
In addition, sample pretreatment is a deproteinization method, and derivatization that has been used in pretreatment for biotin plasma measurement by conventional HPLC is not necessary, so pretreatment can be performed easily and in a short time. it can.
In the conventional HPLC method, in the case of human plasma, the retention time of biotin is 7 to 8 minutes at the earliest, and most of the measurement of one sample takes several tens of minutes to one hour. In the measurement method of the invention, since the analysis cycle of one sample is as short as about 5 minutes (the retention time of biotin is less than 2 minutes), efficient multi-sample processing is possible.
本発明は、生体試料中のビオチンまたはビオチン関連物質を測定する方法であって、
（１）生体試料中のタンパク質を除去する工程と、
（２）工程（１）で得られた試料中のビオチンまたはビオチン関連物質を、高速液体クロマトグラフ／タンデム質量分析法を用いて測定する工程と
を含む、方法に関するものである。
The present invention is a method for measuring biotin or a biotin-related substance in a biological sample,
(1) removing a protein in the biological sample;
(2) The method includes measuring the biotin or biotin-related substance in the sample obtained in step (1) using high performance liquid chromatography / tandem mass spectrometry.
本発明における「生体試料」とは、ヒト、動物、または植物から得られる、尿、血液、血漿、組織、細胞などの材料を含む試料を意味し、本発明の方法により測定可能な試料であれば特に限定されない。なかでも、血漿および尿が好ましく、血漿が特に好ましい。さらに、透析廃液のように、生体由来成分が含まれる試料も本発明の「生体試料」として好適に用いられる。
なかでも、ヒト由来の生体試料が好ましい。ヒトとしては、健常者であっても、種々の疾患を患う患者、例えば、腎不全患者、血液透析患者、血液透析濾過患者、腹膜透析患者、ビオチン欠乏症患者などであってもよいが、ビオチン量の欠乏および過剰による影響を受けやすいことから、特に血液透析患者、血液透析濾過患者、腹膜透析患者が挙げられる。
血漿は、血液に含まれる液体成分の一つで血液の５５％を占める。血清とフィブリノーゲンから成り、物質の輸送、ガス交換、血液凝固、免疫に関与するほか、浸透圧や水素イオン濃度の調節などによって内部環境を整えるのに重要な役割を果たしている。
本発明における「血漿」としては、特に限定されず、ヒト、ラット、マウス、イヌ、サルなどの哺乳動物の血液由来のものが挙げられる。また、その調製方法も特に限定されず、従来既知の方法を用いることができる。好ましくは、採血後、抗凝固剤（ヘパリンナトリウム、ＥＤＴＡ-２Ｎａ、ＥＤＴＡ-２Ｋ等）入りのプラスチックチューブに移し、氷冷化で保存し、４℃で遠心分離後、採取する。なかでも、ヒトの血漿が好ましい。ヒトとしては、健常者、血液透析患者、ビオチン欠乏症患者等が挙げられ、特に血液透析患者が挙げられる。
透析廃液は、血液浄化療法により透析患者血液中の老廃物の除去や電解質濃度の調整等を行った後の透析液であり、透析患者血液中の低分子代謝物等が含まれている。血液透析患者は合併症として貧血症状を有しており、採血は最小限にすることが望ましいとされている。そのため、非侵襲性に採取可能な透析廃液で透析患者の血中代謝物濃度を測定することは患者に負担を与えることがなく、さらに、高分子フィルターを通過した除蛋白試料であることから分析が容易であるといった利点がある。しかしながら、これまで透析廃液中のビオチンを測定した例はなかった。
本発明における「透析廃液」としては、特に限定されず、腎不全患者、血液透析患者、血液透析濾過患者、腹膜透析患者、ビオチン欠乏症患者などの血液浄化療法が必要な患者に対する、血液透析、腹膜透析、血液濾過、血液透析濾過（ＨＤＦ）、持続的血液透析濾過などの血液浄化療法から生じる廃液が挙げられる。
尿は、腎臓において血液が濾過されることにより作られる排泄物であり、体内で生産される老廃物の除去や体内の水分量を調整する働きを持つ。成分のほとんどが水分であり、他に尿素や電解質等を含む。
本発明における「尿」としては、特に限定されず、ヒト、ラット、マウス、イヌ、サルなどの哺乳動物由来のものが挙げられる。なかでも、ヒトの尿が好ましい。ヒトとしては、健常者、排尿が確認される初期の腎不全患者や血液透析患者、血液透析濾過患者、腹膜透析患者、ビオチン欠乏症患者等が挙げられ、特に腎不全患者が挙げられる。
The “biological sample” in the present invention means a sample containing materials such as urine, blood, plasma, tissue, and cells obtained from humans, animals, or plants, and can be measured by the method of the present invention. If it does not specifically limit. Of these, plasma and urine are preferable, and plasma is particularly preferable. Furthermore, a sample containing a biological component such as dialysis waste liquid is also preferably used as the “biological sample” of the present invention.
Of these, a human-derived biological sample is preferable. The human may be a healthy person or a patient suffering from various diseases such as renal failure patient, hemodialysis patient, hemodiafiltration patient, peritoneal dialysis patient, biotin deficiency patient, etc. In particular, hemodialysis patients, hemodiafiltration patients, and peritoneal dialysis patients are included because they are easily affected by deficiency and excess.
Plasma is one of the liquid components contained in blood and accounts for 55% of blood. Consisting of serum and fibrinogen, it plays an important role in regulating the internal environment by regulating osmotic pressure and hydrogen ion concentration in addition to being involved in substance transport, gas exchange, blood coagulation and immunity.
The “plasma” in the present invention is not particularly limited, and examples thereof include those derived from blood of mammals such as humans, rats, mice, dogs and monkeys. Moreover, the preparation method is not particularly limited, and a conventionally known method can be used. Preferably, after blood collection, it is transferred to a plastic tube containing an anticoagulant (heparin sodium, EDTA-2Na, EDTA-2K, etc.), stored on ice, and centrifuged at 4 ° C. and collected. Of these, human plasma is preferred. Examples of humans include healthy individuals, hemodialysis patients, biotin deficiency patients, and the like, and particularly hemodialysis patients.
The dialysis waste liquid is a dialysis liquid after removing waste products in the blood of the dialysis patient and adjusting the electrolyte concentration by blood purification therapy, and includes low-molecular metabolites and the like in the dialysis patient blood. Hemodialysis patients have anemia as a complication and it is desirable to minimize blood collection. Therefore, measuring the metabolite concentration in the blood of a dialysis patient with dialysis waste liquid that can be collected non-invasively does not give a burden to the patient, and it is analyzed because it is a deproteinized sample that has passed through a polymer filter There is an advantage that it is easy. However, there has been no example of measuring biotin in the dialysis waste liquid until now.
The “dialysis waste fluid” in the present invention is not particularly limited, and hemodialysis and peritoneum for patients requiring blood purification therapy such as renal failure patients, hemodialysis patients, hemodiafiltration patients, peritoneal dialysis patients, biotin deficiency patients and the like. Examples include effluents from blood purification therapies such as dialysis, hemofiltration, hemodiafiltration (HDF), continuous hemodiafiltration.
Urine is excretion produced by filtering blood in the kidney, and has the function of removing waste products produced in the body and adjusting the amount of water in the body. Most of the components are moisture, and other components include urea and electrolytes.
The “urine” in the present invention is not particularly limited, and examples thereof include those derived from mammals such as humans, rats, mice, dogs, monkeys and the like. Of these, human urine is preferable. Examples of humans include healthy subjects, early renal failure patients with confirmed urination, hemodialysis patients, hemodiafiltration patients, peritoneal dialysis patients, biotin deficiency patients, and the like, particularly renal failure patients.
ビオチンは、下記の構造式で示される化合物（分子式：Ｃ１０Ｈ１６Ｎ２Ｏ３Ｓ、分子量：２４４．３１）であり、ビタミンＢ群に属する水溶性ビタミンの一種であり、ビタミンＢ７またはビタミンＨとも呼ばれている。
ビオチンの構造式：
また、上記「ビオチン」および「ビオチン関連物質」の各種の塩も本発明の測定法の対象となる。
Biotin is a compound represented by the following structural formula (molecular formula: C 10 H 16 N 2 O 3 S, molecular weight: 244.31), and is a kind of water-soluble vitamin belonging to the vitamin B group, such as vitamin B7 or vitamin Also called H.
Structural formula of biotin:
In addition, various salts of the above “biotin” and “biotin-related substance” are also subject to the measurement method of the present invention.
上記工程（１）は、生体試料中に含まれるタンパク質を除去する工程である。試料中にタンパク質が多量に存在すると、上記工程（２）におけるビオチンまたはビオチン関連物質の測定が困難になるためである。工程（１）としては、特に限定されないが、例えば、タンパク質の変性による不溶化、物理的な除去、浸透制限充填剤の利用などの方法が挙げられる。
「タンパク質の変性による不溶化」としては、例えば、過塩素酸、トリクロロ酢酸、メタリン酸のような酸の添加；アセトン、アセトニトリル、メタノール、エタノールのような有機溶媒の添加；加熱；冷却などの方法が挙げられる。
「物理的な除去」としては、例えば、メンブレンフィルターなどを使用した限外ろ過、透析チューブなどを使用した透析、超遠心分離などが挙げられる。
上記タンパク質を除去方法は、１種の方法のみでも２種以上の方法を組み合わせてもよい。
The step (1) is a step of removing proteins contained in the biological sample. This is because when a large amount of protein is present in the sample, it is difficult to measure biotin or a biotin-related substance in the step (2). Although it does not specifically limit as process (1), For example, methods, such as insolubilization by protein modification | denaturation, a physical removal, and the utilization of a permeation restriction | limiting filler, are mentioned.
Examples of “insolubilization by protein denaturation” include methods such as addition of acids such as perchloric acid, trichloroacetic acid, and metaphosphoric acid; addition of organic solvents such as acetone, acetonitrile, methanol, and ethanol; heating; and cooling. Can be mentioned.
Examples of “physical removal” include ultrafiltration using a membrane filter, dialysis using a dialysis tube, and ultracentrifugation.
The method for removing the protein may be a single method or a combination of two or more methods.
工程（１）としては、タンパク質と結合したビオチンを遊離させる点から、特に、有機溶媒の添加によりタンパク質を不溶化（沈殿）させ、除去することが好ましい。有機溶媒としては、特に、ビオチンの回収率の点から、メタノールが好ましい。
生体試料中に有機溶媒（例えばメタノール）を添加してタンパク質を除去する場合、例えば、以下の手順および条件で行うことが好ましい。
まず、生体試料中に対して所定量のメタノールを混合し、該混合物を所定の時間撹拌する。メタノールの添加量は生体試料の量に基づいて定めることができる。通常、生体試料に対して、メタノールを通常等量〜１００倍量、好ましくは等量〜１０倍量使用するが、生体試料の使用量、除タンパク効率、上清採取量および蒸発乾固後の再溶解量の点から、好ましくは生体試料５０〜５００μＬに対して、メタノールを生体試料の３〜１０倍量使用することができる。撹拌時間および温度は、特に限定されないが、例えば、撹拌時間は１０秒〜１分間、温度は１５〜３０℃とすることができる。その後、遠心分離して沈殿を除き、上清を採取する。遠心分離は、特に限定されないが、例えば、８，０００〜１５，０００×ｇで、４℃〜３０℃、１０〜１５分間の条件で行うことができる。
As the step (1), it is preferable to insolubilize (precipitate) and remove the protein by adding an organic solvent, particularly from the viewpoint of releasing biotin bound to the protein. As the organic solvent, methanol is particularly preferable from the viewpoint of biotin recovery.
When an organic solvent (for example, methanol) is added to a biological sample to remove a protein, it is preferably performed, for example, according to the following procedure and conditions.
First, a predetermined amount of methanol is mixed into a biological sample, and the mixture is stirred for a predetermined time. The amount of methanol added can be determined based on the amount of the biological sample. Usually, methanol is usually used in an equivalent amount to 100 times, preferably 10 to 10 times the amount of the biological sample. However, the amount of biological sample used, the protein removal efficiency, the amount of supernatant collected, and the amount after evaporation to dryness From the viewpoint of the re-dissolution amount, methanol can be used in an
上記工程（２）は、工程（１）で得られたタンパク質除去後の試料中のビオチンまたはビオチン関連物質を、高速液体クロマトグラフ／タンデム質量分析法（以下、「ＬＣ／ＭＳ／ＭＳ」と称する）を用いて測定する工程である。
ＬＣ／ＭＳ／ＭＳは、試料をＨＰＬＣで分離し、溶出成分をイオン化室に導入し、イオン化してマススペクトルを得、これを第１段目の質量分析計（ＭＳ）でイオン（分子イオンの付加体）を選択し、コリジョンセルと呼ばれる衝突室で不活性ガスを衝突させてイオンを解離させて、第２段目のＭＳで分析する方法である。
In the step (2), biotin or a biotin-related substance in the sample after protein removal obtained in the step (1) is referred to as high performance liquid chromatograph / tandem mass spectrometry (hereinafter referred to as “LC / MS / MS”). ) To measure.
In LC / MS / MS, a sample is separated by HPLC, an elution component is introduced into an ionization chamber, and ionized to obtain a mass spectrum, which is ionized by a first-stage mass spectrometer (MS) (of molecular ions). This is a method in which an adduct is selected, an inert gas is collided in a collision chamber called a collision cell, ions are dissociated, and analysis is performed by the second-stage MS.
本発明におけるＬＣ／ＭＳ／ＭＳに使用する機器としては、特に限定されず、例えば、液体クロマトグラフ用の機器としては、Ｎｅｘｅｒａ（島津製作所製）、ＡＣＱＵＩＴＹ ＵＰＬＣ（ウォーターズ製）などが挙げられ、タンデム質量分析用の機器としては、例えば、ＱＴＲＡＰ ５５００（エービー・サイエックス製）、ＴＳＱ Ｖａｎｔａｇｅ（サーモフィッシャーサイエンティフィック製）などが挙げられる。 The instrument used for LC / MS / MS in the present invention is not particularly limited. Examples of the instrument for liquid chromatography include Nexera (manufactured by Shimadzu Corporation), ACQUITY UPLC (manufactured by Waters), and the like. Examples of the instrument for mass spectrometry include QTRAP 5500 (manufactured by AB Scientific), TSQ Vantage (manufactured by Thermo Fisher Scientific), and the like.
上記工程（２）におけるＨＰＬＣ用のカラムとしては、例えば、疎水性相互作用、双極子-双極子相互作用およびπ-π相互作用から選択される少なくとも１種の作用により分離するカラムが挙げられる。
疎水性相互作用により分離するカラムとしては、例えば、カラム担体の官能基としてオクタデシル基を含むカラム（Ｃ１８カラム）、具体的には、ＡＣＱＵＩＴＹ ＵＰＬＣＴＭ ＨＳＳ Ｔ３（ウォーターズ製）、ＹＭＣ―Ｔｒｉａｒｔ Ｃ１８（ワイエムシィ製）などが挙げられる。
双極子-双極子相互作用により分離するカラムとしては、例えば、カラム担体の官能基としてフッ素が結合した直鎖または分岐鎖のアルキル基（例えば炭素数３〜９個、特に炭素数３個）を含むカラム、具体的には、Ｆｌｕｏｆｉｘ-ＩＩ １２０Ｅ（和光純薬工業製）、Ｆｌｕｏｐｈａｓｅ ＲＰ（サーモフィッシャーサイエンティフィック製）、Ｆｌｕｏｐｈａｓｅ ＷＰ（サーモフィッシャーサイエンティフィック製）などが挙げられる。
π-π相互作用により分離するカラムとしては、例えば、カラム担体の官能基として芳香環基（フェニル基、ビフェニル基など）を含むカラム、具体的には、Ｘｓｅｌｅｃｔ ＣＳＨ Ｆｌｕｏｒｏ―Ｐｈｅｎｙｌカラム（ウォーターズ製）、Ａｌｌｕｒｅ Ｂｉｐｈｅｎｙｌ カラム（レステック製）、Ｉｎｅｒｔｓｉｌ Ｐｈ―３（ジーエルサイエンス製）、ＹＭＣ―Ｐａｃｋ Ｐｈ（ワイエムシィ製）、ＸＢｒｉｄｇｅ ＢＥＨ Ｐｈｅｎｙｌ （ウォーターズ製）などが挙げられる。
疎水性相互作用、双極子-双極子相互作用およびπ-π相互作用により分離するカラムとしては、例えば、カラム担体の官能基としてペンタフルオロフェニル基が結合した直鎖または分岐鎖のアルキル基（例えば炭素数３〜９個、特に炭素数３個）を含むカラム、具体的には、Ａｓｃｅｎｔｉｓ Ｅｘｐｒｅｓｓ Ｆ５（シグマ-アルドリッチ製）、ＴＣＩ Ｓｔｅｌｌａ ＰＦＰ（東京化成製）、Ｋｉｎｅｔｅｘ２．６μｍミニボアカラム（フェノメネクス製）、ＣＡＰＣＥＬＬ ＣＯＲＥ（資生堂製）、Ｐｕｒｓｕｉｔ ＰＦＰ（アジレント・テクノロジー製）、ＸＳｅｌｅｃｔ ＨＳＳ ＰＦＰ ＸＰ Ｃｏｌｕｍｎ（ウォーターズ製）、ＹＭＣ―Ｔｒｉａｒｔ ＰＦＰ（ワイエムシィ製）、Ｈｙｐｅｒｓｉｌ ＧＯＬＤ ＰＦＰ（サーモフィッシャーサイエンティフィック製）などが挙げられる。
本発明においては、ビオチンおよびビオチン関連物質と他の成分とを明確に分離することができることから、疎水性相互作用、双極子-双極子相互作用およびπ-π相互作用により分離するカラムが好ましく、特にカラム担体の官能基としてペンタフルオロフェニル基が結合した直鎖または分岐鎖のアルキル基（例えば炭素数３〜９個、特に炭素数３個）を含むカラムが好ましい。
Examples of the HPLC column in the step (2) include a column that is separated by at least one action selected from a hydrophobic interaction, a dipole-dipole interaction, and a π-π interaction.
As a column separated by hydrophobic interaction, for example, a column containing an octadecyl group as a functional group of a column carrier (C18 column), specifically, ACQUITY UPLC ™ HSS T3 (manufactured by Waters), YMC-Triart C18 (YMC) Manufactured).
As a column to be separated by dipole-dipole interaction, for example, a linear or branched alkyl group (for example, having 3 to 9 carbon atoms, particularly 3 carbon atoms) bonded with fluorine as a functional group of the column carrier is used. Examples of the column include Fluofix-II 120E (manufactured by Wako Pure Chemical Industries), Fluophase RP (manufactured by Thermo Fisher Scientific), Fluophase WP (manufactured by Thermo Fisher Scientific), and the like.
As a column separated by π-π interaction, for example, a column containing an aromatic ring group (phenyl group, biphenyl group, etc.) as a functional group of the column carrier, specifically, an Xselect CSH Fluoro-Phenyl column (manufactured by Waters) Allure Biphenyl column (manufactured by Restec), Inertsil Ph-3 (manufactured by GL Science), YMC-Pack Ph (manufactured by YMC), XBridge BEH Phenyl (manufactured by Waters), and the like.
As a column separated by hydrophobic interaction, dipole-dipole interaction and π-π interaction, for example, a linear or branched alkyl group to which a pentafluorophenyl group is bonded as a functional group of the column carrier (for example, A column containing 3 to 9 carbon atoms, particularly 3 carbon atoms, specifically, Ascentis Express F5 (manufactured by Sigma-Aldrich), TCI Stella PFP (manufactured by Tokyo Chemical Industry), Kintex 2.6 μm minibore column (manufactured by Phenomenex), CAPCELL CORE (manufactured by Shiseido), Pursuit PFP (manufactured by Agilent Technologies), XSelect HSS PFP XP Column (manufactured by Waters), YMC-Triart PFP (manufactured by YMC), Hypersil GOLD PFP (thermo fisher sien Tiffic).
In the present invention, since biotin and biotin-related substances and other components can be clearly separated, a column that separates by hydrophobic interaction, dipole-dipole interaction and π-π interaction is preferable. In particular, a column containing a linear or branched alkyl group (for example, having 3 to 9 carbon atoms, particularly 3 carbon atoms) to which a pentafluorophenyl group is bonded as a functional group of the column carrier is preferable.
ＨＰＬＣの条件は、特に限定されず、使用するカラムに応じて、移動相、流速、カラム温度、などを決定すればよい。
例えばカラム担体の官能基としてペンタフルオロフェニル基が結合した直鎖または分岐鎖のアルキル基（例えば炭素数３〜９個、特に炭素数３個）を含むカラムを使用する場合、例えば以下の条件で行うことが好ましい。
移動相としては、ギ酸、酢酸等の揮発性の酸および／またはギ酸アンモニウムや酢酸アンモニウムなど揮発性の塩を用いて、ｐＨを２〜８に調整した溶液と、メタノールやアセトニトリルなどの有機溶媒との混液を使用することができる。カラム温度および流速並びに試料注入量は特に限定されないが、カラムや装置の性能に合わせて、カラム温度は２０〜５０℃、流速は０．２〜０．６ｍＬ/ｍｉｎ、試料注入量は１〜２０μＬの条件で行うことができる。
The HPLC conditions are not particularly limited, and the mobile phase, flow rate, column temperature, etc. may be determined according to the column used.
For example, when using a column containing a linear or branched alkyl group (for example, 3 to 9 carbon atoms, particularly 3 carbon atoms) to which a pentafluorophenyl group is bonded as a functional group of the column carrier, for example, under the following conditions: Preferably it is done.
As a mobile phase, a volatile acid such as formic acid and acetic acid and / or a volatile salt such as ammonium formate and ammonium acetate and a pH adjusted to 2 to 8; an organic solvent such as methanol and acetonitrile; Can be used. The column temperature and flow rate, and the sample injection amount are not particularly limited, but the column temperature is 20 to 50 ° C., the flow rate is 0.2 to 0.6 mL / min, and the sample injection amount is 1 to 20 μL according to the performance of the column and the apparatus. Can be performed under the following conditions.
本発明の測定法は、上記工程（１）および（２）の二工程のみを必須工程とするものであり、従来のＨＰＬＣによるビオチン血漿測定法の前処理において使用されていた誘導体化などの工程が不要であるので、生体試料中のビオチンおよびビオチン関連物質を迅速かつ簡便に高感度で測定することができる。本発明の測定法では、１検体の分析サイクルは、通常、５分程度である。したがって、効率的な多検体処理が可能であり、健康管理や疾患の診断、ビオチンの体内動態に関する研究において、有利に使用することができる。 The measurement method of the present invention comprises only the above-mentioned steps (1) and (2) as essential steps, and the steps such as derivatization used in the pretreatment of the conventional biotin plasma measurement method by HPLC. Therefore, biotin and biotin-related substances in a biological sample can be measured quickly and easily with high sensitivity. In the measurement method of the present invention, the analysis cycle of one specimen is usually about 5 minutes. Therefore, efficient multi-sample processing is possible, and it can be advantageously used in research on health care, diagnosis of diseases, and biokinetics of biotin.
本発明はまた、生体試料中のビオチンまたはビオチン関連物質を測定するための測定用キットであって、
（１）生体試料中のタンパク質を除去する工程に使用するための試薬と、
（２）高速液体クロマトグラフ／タンデム質量分析法に使用するための機器
を含む、キットに関する。
当該キットに使用するための試薬（１）および機器（２）としては、上記本発明の測定法に使用する試薬および機器が挙げられる。
The present invention also provides a measurement kit for measuring biotin or a biotin-related substance in a biological sample,
(1) a reagent for use in the step of removing proteins in a biological sample;
(2) It relates to a kit including an instrument for use in high performance liquid chromatograph / tandem mass spectrometry.
Examples of the reagent (1) and the instrument (2) for use in the kit include the reagent and instrument used in the measurement method of the present invention.
以下、本発明の測定法を、実施例を参照して説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, although the measuring method of this invention is demonstrated with reference to an Example, this invention is not limited to these Examples.
実施例１
１. 標準溶液および内標準溶液の調製
１．１ 標準原液
ビオチン（分子式：Ｃ１０Ｈ１６Ｎ２Ｏ３Ｓ、分子量：２４４．３１、サンケミカル株式会社製）１０ｍｇを正確に量り、メタノールでメスフラスコにて１０ｍＬとし超音波洗浄器で溶解して１ｍｇ/ｍＬの標準原液を調製した。
ビオチンの構造式：
１．２ 検量線用標準溶液
標準原液を下表１に従いシリコンコーティングされたガラス試験管を用いてメタノールで希釈し、検量線用標準溶液を調製した。
Example 1
1. Preparation of standard solution and internal standard solution 1.1 Standard stock solution Biotin (molecular formula: C 10 H 16 N 2 O 3 S, molecular weight: 244.31, manufactured by Sun Chemical Co., Ltd.) is accurately weighed and measured in methanol with methanol. A standard stock solution of 1 mg / mL was prepared by dissolving with an ultrasonic cleaner to 10 mL in a flask.
Structural formula of biotin:
1.2 Standard Solution for Calibration Curve A standard stock solution was diluted with methanol using a silicon-coated glass test tube according to Table 1 below to prepare a calibration curve standard solution.
１．３． 内標準原液および内標準溶液
ｒａｃ ビオチン-ｄ４（分子式：Ｃ１０Ｈ１２Ｄ４Ｎ２Ｏ３Ｓ、分子量：２４８．３４、トロントリサーチケミカルズ製）の１ｍｇ入り製品の容器にメタノールを１ｍＬ添加し、超音波洗浄器で溶解して１ｍｇ／ｍＬの内標準原液を調製した。
ｒａｃ ビオチン-ｄ４の構造式：
この内標準原液２００μＬを２０ｍＬのメスフラスコに採取して、メタノールで２０ｍＬにメスアップし、１０μｇ／ｍＬの内標準溶液（ＩＳＳ-１）を調製した。
ＩＳＳ-１を下表２に従いメスフラスコを用いてメタノールで希釈し、内標準溶液（ＩＳＳ-３）を調製した。なお、調製量の増減は可とし、必要量に応じて同比率にて調製した。
1.3. Add 1 mL of methanol to a 1 mg product container of internal standard stock solution and internal standard solution rac biotin-d4 (molecular formula: C 10 H 12 D 4 N 2 O 3 S, molecular weight: 248.34, manufactured by Toronto Research Chemicals) An internal standard stock solution of 1 mg / mL was prepared by dissolving with an ultrasonic cleaner.
rac biotin-d4 structural formula:
200 μL of this internal standard stock solution was collected in a 20 mL volumetric flask and made up to 20 mL with methanol to prepare a 10 μg / mL internal standard solution (ISS-1).
According to Table 2 below, ISS-1 was diluted with methanol using a volumetric flask to prepare an internal standard solution (ISS-3). It should be noted that the amount of preparation could be increased or decreased, and the amount was adjusted according to the required amount.
２. 分析法検証用試料の調製
２．１． 検量線用試料
検量線用標準溶液ＳＳ-５〜ＳＳ-１０を３０μＬずつ採取し、Ｄ-ＰＢＳ（-）（細胞培養用、和光純薬工業製） ３００μＬを添加し、検量線用試料を調製した（調製濃度２、１、０．５、０．４、０．２および０．１ｎｇ／ｍＬ）。
ブランク試料およびゼロ試料はメタノール３０μＬにＤ-ＰＢＳ（-）３００μＬを添加した。
２．２. 血漿試料
正常ヒト血漿・プール（コスモバイオ製、抗凝固剤：ヘパリンナトリウム、以下、「ヒト血漿」と称する）３００μＬにメタノール３０μＬを添加し、血漿試料を調製した。
２．３. ビオチン添加血漿試料
検量線用標準溶液ＳＳ-４（１００ｎｇ／Ｌ）をメタノールで２倍に希釈して５０ｎｇ/ｍＬ溶液を調製後、１０ｍＬのメスフラスコに１００μＬ採取し、ヒト血漿で正確にメスアップしたビオチン濃度０．５ｎｇ／ｍＬの溶液３００μＬにメタノールを３０μＬを添加し、ビオチン添加血漿試料を調製した。
2. Preparation of analytical method verification sample 2.1. Sample for calibration curve Collect 30 μL each of standard solutions SS-5 to SS-10 for calibration curve, add 300 μL of D-PBS (-) (for cell culture, manufactured by Wako Pure Chemical Industries), and prepare a sample for calibration curve (
As for the blank sample and the zero sample, 300 μL of D-PBS (−) was added to 30 μL of methanol.
2.2. Plasma Sample 30 μL of methanol was added to 300 μL of normal human plasma / pool (manufactured by Cosmo Bio, anticoagulant: heparin sodium, hereinafter referred to as “human plasma”) to prepare a plasma sample.
2.3. Biotin-added plasma sample A calibration curve standard solution SS-4 (100 ng / L) was diluted 2-fold with methanol to prepare a 50 ng / mL solution, and then 100 μL was collected in a 10 mL volumetric flask. 30 μL of methanol was added to 300 μL of a biotin concentration of 0.5 ng / mL that was accurately measured to prepare a biotin-added plasma sample.
３. 試料前処理法
以下の手順で試料の前処理を行った。
（１）検量線用試料、ゼロ試料およびビオチン添加血漿試料には内標準溶液ＩＳＳ-３を３０μＬ添加した。ブランク試料および血漿試料にはメタノールを３０μＬ添加した。
（２）各試料にメタノールを１．２ｍＬ添加し、血漿を含まない試料は約５〜１０秒間、血漿を含む試料は約１分間撹拌した。
（３）血漿を含まない試料は１ｍＬを採取し、血漿を含む試料は４℃、１０，０００ｒｐｍ（９１７５×ｇ）で１０分間遠心し、上清を１ｍＬ採取した。
（４）５０℃で窒素ガスによる蒸発乾固を行い、残渣に１０％メタノール２００μＬを添加し、超音波洗浄器で１５分間溶解した。
（５）溶解後、ＨＰＬＣバイアルに移し、血漿を含まない試料はそのままＬＣ/ＭＳ/ＭＳで分析した。血漿を含む試料は０．２μｍのフィルター（マイレクス-ＬＨフィルターユニット、フィルター直径４ｍｍ、フィルター孔径０．２０μｍ、ＰＴＦＥ、親水性、ミリポア製）でろ過後、ＨＰＬＣバイアルに移し、ＬＣ/ＭＳ/ＭＳで分析した。
3. Sample pretreatment The sample was pretreated by the following procedure.
(1) 30 μL of the internal standard solution ISS-3 was added to the calibration curve sample, zero sample, and biotin-added plasma sample. 30 μL of methanol was added to the blank sample and the plasma sample.
(2) 1.2 mL of methanol was added to each sample, the sample not containing plasma was stirred for about 5 to 10 seconds, and the sample containing plasma was stirred for about 1 minute.
(3) 1 mL of the sample containing no plasma was collected, and the sample containing plasma was centrifuged at 10,000 rpm (9175 × g) for 10 minutes at 4 ° C., and 1 mL of the supernatant was collected.
(4) Evaporation to dryness was performed with nitrogen gas at 50 ° C., 200 μL of 10% methanol was added to the residue, and dissolved in an ultrasonic cleaner for 15 minutes.
(5) After dissolution, the sample was transferred to an HPLC vial, and the sample containing no plasma was directly analyzed by LC / MS / MS. Samples containing plasma were filtered through a 0.2 μm filter (Mirex-LH filter unit, filter
４. 分析条件
分析には以下の機器を使用した。
4. Analysis conditions The following equipment was used for analysis.
４．１. ＨＰＬＣの移動相の調製
ギ酸アンモニウム６３０．６ｍｇを正確に量り、精製水でメスフラスコにて１０００ｍＬとし、１０ｍｍｏｌ／Ｌギ酸アンモニウム溶液を調製した。これにギ酸を添加してｐＨ３．０に調整して移動相Ａ液とした。
４．２. ＨＰＬＣ条件
分析カラム：ACQUITY UPLC HSS T3 (1.8μm、2.1×100mm)（ウォーターズ製）
ガードフィルター：KrudKatcher Ultra In-Line Filter（フェノメネクス製）
カラム温度：50℃
移動相：A液：10mmol/Lギ酸アンモニウム（pH 3.0）、B液：アセトニトリル
タイムプログラム：
0→1.5min：20%B
1.5→3.0min：80%B
3.0→5.0min：20%B（分析サイクル：5分）
流速：0.4mL/min
４．３. オートサンプラー条件
オートサンプラー設定温度：4℃
洗浄溶媒：10％メタノール
注入量：15μL
４．４. ＭＳ/ＭＳ条件
イオン化法：ESI法（ポジティブイオンモード）
イオンスプレイ電圧：5500V
ターボヒーター：700℃
カーテンガス：30psi
ネブライザーガス：30psi
デソルベーションガス：80psi
測定法：MRM
モニターイオン：
ビオチン：m/z 245＞227（CE19）
rac biotin-d4：m/z 249＞231（CE19）
4.1. Preparation of HPLC mobile phase 630.6 mg of ammonium formate was accurately weighed and made up to 1000 mL in a volumetric flask with purified water to prepare a 10 mmol / L ammonium formate solution. To this was added formic acid to adjust the pH to 3.0 to obtain mobile phase A solution.
4.2. HPLC conditions Analysis column: ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 100 mm) (manufactured by Waters)
Guard filter: KrudKatcher Ultra In-Line Filter (Phenomenex)
Column temperature: 50 ° C
Mobile phase: Liquid A: 10 mmol / L ammonium formate (pH 3.0), Liquid B: acetonitrile Time program:
0 → 1.5min: 20% B
1.5 → 3.0min: 80% B
3.0 → 5.0min: 20% B (analysis cycle: 5 minutes)
Flow rate: 0.4mL / min
4.3. Autosampler conditions Autosampler set temperature: 4 ° C
Washing solvent: 10% methanol Injection volume: 15μL
4.4. MS / MS conditions Ionization method: ESI method (positive ion mode)
Ion spray voltage: 5500V
Turbo heater: 700 ℃
Curtain gas: 30psi
Nebulizer gas: 30psi
Desolvation gas: 80psi
Measurement method: MRM
Monitor ion:
Biotin: m / z 245> 227 (CE19)
rac biotin-d4: m / z 249> 231 (CE19)
５. 濃度算出法
検量線用試料（６濃度：０．１、０．２、０．４、０．５、１および２ｎｇ／ｍＬ、各濃度ｎ＝１）を調製し、測定した。測定には内標準物質（ＩＳＳ-３）とのピーク面積比による内標準法により行った。定量値は、Ａｎａｌｙｓｔ Ｖｅｒ．１．６．１（エービー・サイエックス製）により算出した。検量線の重み付けには１／ｘ２を用いた。
5. Concentration calculation method Calibration curve samples (6 concentrations: 0.1, 0.2, 0.4, 0.5, 1 and 2 ng / mL, each concentration n = 1) were prepared and measured. The measurement was performed by the internal standard method based on the peak area ratio with the internal standard substance (ISS-3). Quantitative values were analyzed using Analyst Ver. It was calculated according to 1.6.1 (manufactured by AB SciX). The weighting of the calibration curve using 1 / x 2.
６. 結果
６．１. 血漿試料
図１に血漿試料および検量線試料（０．０５ｎｇ／ｍＬ）のクロマトグラムを示す。内標準物質について、血漿中に妨害と成り得るピークは確認できなかった。
６．２. 検量線
図２に典型的な検量線の結果を示す。０．１〜２ｎｇ／ｍＬの濃度範囲において、検量線の直線性は３回の測定において相関係数（ｒ）がそれぞれ０．９９９６、０．９９９６および０．９９９８、相対誤差（％ＲＥ）はそれぞれ-２．０〜＋３．０％、-２．８〜＋２．４％、-２．３〜＋１．５％であり、判断基準（ｒ：０．９９以上、％ＲＥ：定量下限濃度で±２０％以内、その他で±１５％以内）を満たしていた。また、定量限界値は０．１ｎｇ／ｍＬであった。
６．３. ビオチンおよびｒａｃ ビオチン-ｄ４添加血漿試料
図４にビオチンおよびｒａｃビオチン-ｄ４添加血漿試料のクロマトグラムを示す。
また、表４に得られたビオチン添加血漿試料の定量値から血漿試料（ブランク）の定量値の平均値を差し引き、真度（％ＲＥ）、精度（％ＣＶ）および回収率（％）を下記式（１）〜（３）により算出した結果を示した。０．５ｎｇ／ｍＬ添加試料において、精度（％ＣＶ）は２．２％、真度（％ＲＥ）は-３．４％であり、判断基準（％ＣＶ：１５．０％以下、％ＲＥ：±１５．０％以内）を満たしていた。
6.2. Calibration curve Fig. 2 shows the results of a typical calibration curve. In the concentration range of 0.1 to 2 ng / mL, the linearity of the calibration curve is 0.9996, 0.9996, and 0.9998, respectively, and the relative error (% RE) is 3 times in three measurements. -2.0 to + 3.0%, -2.8 to + 2.4%, and -2.3 to + 1.5%, respectively. Judgment criteria (r: 0.99 or more,% RE: lower limit of quantification) Within ± 20%, and within ± 15% in other cases). Moreover, the quantitative limit value was 0.1 ng / mL.
6.3. Plasma samples with biotin and rac biotin-d4 added Figure 4 shows chromatograms of plasma samples with biotin and rac biotin-d4 added.
In addition, the average value of the quantitative value of the plasma sample (blank) is subtracted from the quantitative value of the biotin-added plasma sample obtained in Table 4, and the accuracy (% RE), accuracy (% CV), and recovery rate (%) are shown below. The results calculated by equations (1) to (3) are shown. In the sample added with 0.5 ng / mL, the accuracy (% CV) is 2.2% and the accuracy (% RE) is −3.4%, and the judgment criteria (% CV: 15.0% or less,% RE: Within ± 15.0%).
実施例２
１. 標準溶液および内標準溶液の調製
１．１. 標準原液
ビオチン１０ｍｇを正確に量り、メタノールでメスフラスコにて１０ｍＬとし超音波洗浄器で溶解して１ｍｇ／ｍＬの標準原液を調製した。
１．２. 検量線用標準溶液
標準原液を下表５に従いシリコンコーティングされたガラス試験管を用いてメタノールで希釈し、検量線用標準溶液を調製した。
Example 2
1. Preparation of Standard Solution and Internal Standard Solution 1.1.
1.2. Standard solution for calibration curve The standard stock solution was diluted with methanol using a silicon-coated glass test tube according to Table 5 below to prepare a calibration curve standard solution.
１．３. 内標準原液および内標準溶液
実施例１で調製した内標準溶液（ＩＳＳ-１）２００μＬを５ｍＬのメスフラスコに採取して、メタノールで５ｍＬにメスアップし、４００ｎｇ／ｍＬの内標準溶液（ＩＳＳ-４）を調製した。
1.3. Internal standard stock solution and internal
２. 分析法検証用試料の調製
２．１. 検量線用試料
検量線用標準溶液ＳＳ-５〜ＳＳ-７およびＳＳ-９〜ＳＳ-１１を３０μＬずつ採取し、Ｄ-ＰＢＳ（-）３００μＬを添加し、検量線用試料を調製した（調製濃度２、１、０．５、０．２、０．１および０．０５ｎｇ／ｍＬ）。
ブランク試料およびゼロ試料はメタノール３０μＬにＤ-ＰＢＳ（-）３００μＬを添加した。
２．２. 血漿試料
ヒト血漿３００μＬにメタノール３０μＬを添加し、血漿試料を調製した。
２．３. ビオチン添加血漿試料
検量線用標準溶液ＳＳ-４（１００ｎｇ／Ｌ）をメタノールで２倍に希釈して５０ｎｇ／ｍＬ溶液を調製し、１０ｍＬのメスフラスコに１００μＬ採取し、ヒト血漿で正確にメスアップしたビオチン濃度０．５ｎｇ／ｍＬの溶液３００μＬにメタノールを３０μＬ添加し、ビオチン添加血漿試料を調製した。
2. Preparation of sample for verification of analytical method 2.1. Sample for calibration curve 30 μL each of standard solution for calibration curve SS-5 to SS-7 and SS-9 to SS-11 was collected and 300 μL of D-PBS (−) Were added to prepare calibration curve samples (preparation concentrations of 2, 1, 0.5, 0.2, 0.1 and 0.05 ng / mL).
As for the blank sample and the zero sample, 300 μL of D-PBS (−) was added to 30 μL of methanol.
2.2. Plasma Sample A plasma sample was prepared by adding 30 μL of methanol to 300 μL of human plasma.
2.3. Biotin-added plasma sample A standard solution for calibration curve SS-4 (100 ng / L) was diluted 2-fold with methanol to prepare a 50 ng / mL solution, and 100 μL was collected in a 10-mL volumetric flask. 30 μL of methanol was added to 300 μL of a biotin concentration of 0.5 ng / mL that had been accurately measured to prepare a biotin-added plasma sample.
３. 試料前処理法
以下の手順で試料の前処理を行った。
（１）検量線用試料、ゼロ試料およびビオチン添加血漿試料には内標準溶液ＩＳＳ-４を３０μＬ添加した。ブランク試料および血漿試料にはメタノールを３０μＬ添加した。
（２）各試料にメタノールを１．２ｍＬ添加し、血漿を含まない試料は約５〜１０秒間、血漿を含む試料は約１分間撹拌した。
（３）血漿を含まない試料は１ｍＬを採取し、血漿を含む試料は４℃、１０，０００ｒｐｍで１０分間遠心し、上清を１ｍＬ採取した。
（４）５０℃で窒素ガスによる蒸発乾固を行い、残渣に１％ギ酸/メタノール（９/１）２００μＬを添加し、超音波洗浄器で１５分間溶解した。
（５）溶解後、ＨＰＬＣバイアルに移し、血漿を含まない試料はそのままＬＣ／ＭＳ／ＭＳで分析した。血漿を含む試料は０．４５μｍのフィルター（マイレクス-ＬＨフィルターユニット、フィルター直径４ｍｍ、フィルター孔径０．４５μｍ、ＰＴＦＥ、親水性、ミリポア製）でろ過し、ＨＰＬＣバイアルに移し、ＬＣ/ＭＳ/ＭＳで分析した。
3. Sample pretreatment The sample was pretreated by the following procedure.
(1) 30 μL of the internal standard solution ISS-4 was added to the calibration curve sample, zero sample, and biotin-added plasma sample. 30 μL of methanol was added to the blank sample and the plasma sample.
(2) 1.2 mL of methanol was added to each sample, the sample not containing plasma was stirred for about 5 to 10 seconds, and the sample containing plasma was stirred for about 1 minute.
(3) 1 mL of the sample not containing plasma was collected, and the sample containing plasma was centrifuged at 10,000 rpm for 10 minutes at 4 ° C., and 1 mL of the supernatant was collected.
(4) Evaporation to dryness was performed with nitrogen gas at 50 ° C., and 200 μL of 1% formic acid / methanol (9/1) was added to the residue, and dissolved in an ultrasonic cleaner for 15 minutes.
(5) After dissolution, the sample was transferred to an HPLC vial, and the sample containing no plasma was directly analyzed by LC / MS / MS. Samples containing plasma were filtered with a 0.45 μm filter (Mirex-LH filter unit, filter
４. 分析条件
分析には以下の機器を使用した。
4. Analysis conditions The following equipment was used for analysis.
４．１. ＨＰＬＣの移動相の調製
ギ酸アンモニウム６３０．６ｍｇを正確に量り、精製水でメスフラスコにて１０００ｍＬとし、１０ｍｍｏｌ／Ｌギ酸アンモニウム溶液を調製した。また、ギ酸５００μＬをメスフラスコを用いて精製水で１０ｍＬとして、５％ギ酸溶液を調製した。ギ酸アンモニウム溶液１０００ｍＬに５％ギ酸溶液を２．８ｍＬ添加して撹拌し、ｐＨが約４．１であることを確認し、これを移動相Ａ液とした。
４．２. ＨＰＬＣ条件
分析カラム：Ascentis Express F5 HPLC Column (2.7μm、2.1×100mm)（シグマ-アルドリッチ製）
ガードフィルター：KrudKatcher Ultra In-Line Filter（フェノメネクス製）
カラム温度：50℃
移動相：A液：10mmol/Lギ酸アンモニウム（pH4.1）、B液：アセトニトリル
タイムプログラム：
0→2.5min：7%B
2.5→4.0min：70%B
4.0→5.0min：7%B（分析サイクル：5分）
流速：0.6mL/min
４．３. オートサンプラー条件
オートサンプラー設定温度：4℃
洗浄溶媒：10%メタノール
注入量：20μL
４．４. ＭＳ/ＭＳ条件
イオン化法：ESI法（ポジティブイオンモード）
イオンスプレイ電圧：5500V
ターボヒーター：700℃
カーテンガス：30psi
ネブライザーガス：30psi
デソルベーションガス：80psi
測定法：MRM
モニターイオン：
ビオチン：m/z 245 > 227 (CE19)
rac ビオチン-d4：m/z 249 > 231 (CE19)
4.1. Preparation of HPLC mobile phase 630.6 mg of ammonium formate was accurately weighed and made up to 1000 mL in a volumetric flask with purified water to prepare a 10 mmol / L ammonium formate solution. Moreover, 5% formic acid solution was prepared by making 500 mL of
4.2. HPLC conditions Analysis column: Ascentis Express F5 HPLC Column (2.7 μm, 2.1 × 100 mm) (manufactured by Sigma-Aldrich)
Guard filter: KrudKatcher Ultra In-Line Filter (Phenomenex)
Column temperature: 50 ° C
Mobile phase: Solution A: 10 mmol / L ammonium formate (pH 4.1), Solution B: acetonitrile Time program:
0 → 2.5min: 7% B
2.5 → 4.0min: 70% B
4.0 → 5.0min: 7% B (analysis cycle: 5 minutes)
Flow rate: 0.6mL / min
4.3. Autosampler conditions Autosampler set temperature: 4 ° C
Washing solvent: 10% methanol Injection volume: 20μL
4.4. MS / MS conditions Ionization method: ESI method (positive ion mode)
Ion spray voltage: 5500V
Turbo heater: 700 ℃
Curtain gas: 30psi
Nebulizer gas: 30psi
Desolvation gas: 80psi
Measurement method: MRM
Monitor ion:
Biotin: m / z 245> 227 (CE19)
rac biotin-d4: m / z 249> 231 (CE19)
５. 濃度算出法
検量線用試料（６濃度：０．０５、０．１、０．２、０．５、１および２ｎｇ／ｍＬ、各濃度ｎ＝１）を調製し、測定した。測定には内標準物質（ＩＳＳ-４）とのピーク面積比による内標準法により行った。定量値は、Ａｎａｌｙｓｔ Ｖｅｒ.１.６．１（エービー・サイエックス）により算出した。検量線の重み付けには１／ｘ２を用いた。
5. Concentration Calculation Method Samples for calibration curves (6 concentrations: 0.05, 0.1, 0.2, 0.5, 1 and 2 ng / mL, each concentration n = 1) were prepared and measured. The measurement was performed by the internal standard method based on the peak area ratio with the internal standard substance (ISS-4). The quantitative value was calculated by Analyst Ver. 1.6.1 (Abbex). The weighting of the calibration curve using 1 / x 2.
６. 結果
６．１. 血漿試料
図４に血漿試料および検量線試料（０．１ｎｇ／ｍＬ）のクロマトグラムを示した。内標準物質について、血漿中に妨害と成り得るピークは確認できなかった。
６．２. 検量線
図５に典型的な検量線の結果を示した。０．０５〜２ｎｇ／ｍＬの濃度範囲において、検量線の直線性は３回の測定において相関係数（ｒ）がそれぞれ０．９９９７、０．９９９３および０．９９９７、相対誤差（％ＲＥ）はそれぞれ-４．０〜＋２．０％、-４．０〜＋３.２％、-３．５〜＋２．３％であり、判断基準（ｒ：０．９９以上、％ＲＥ：定量下限濃度で±２０％以内、その他で±１５％以内）を満たしていた。また、定量限界値は０．０５ｎｇ／ｍＬであった。
６．３. ビオチンおよびｒａｃビオチン-ｄ４添加血漿試料
図６にビオチンおよびｒａｃビオチン-ｄ４添加血漿試料のクロマトグラムを示す。
また、表７に得られたビオチン添加血漿試料の定量値から血漿試料（ブランク）の定量値の平均値を差し引き、真度（％ＲＥ）、精度（％ＣＶ）および回収率（％）を上記式（１）〜（３）により算出した結果を示した。０．５ｎｇ／ｍＬ添加試料において、精度（％ＣＶ）は２．２％、真度（％ＲＥ）は-３．４％であり、判断基準（％ＣＶ：１５．０％以下、％ＲＥ：±１５．０％以内）を満たしていた。
6. Results 6.1. Plasma Sample FIG. 4 shows chromatograms of a plasma sample and a calibration curve sample (0.1 ng / mL). For the internal standard, no peak that could interfere with the plasma was confirmed.
6.2. Calibration curve Fig. 5 shows the results of a typical calibration curve. In the concentration range of 0.05 to 2 ng / mL, the linearity of the calibration curve is 0.9997, 0.9993, and 0.9997 for the correlation coefficient (r) in three measurements, and the relative error (% RE) is -4.0 to + 2.0%, -4.0 to + 3.2%, and -3.5 to + 2.3%, respectively. Judgment criteria (r: 0.99 or more,% RE: lower limit of quantification) Within ± 20%, and within ± 15% in other cases). The quantitative limit value was 0.05 ng / mL.
6.3. Plasma sample with biotin and rac biotin-d4 added FIG. 6 shows a chromatogram of the plasma sample with biotin and rac biotin-d4 added.
In addition, the average value of the plasma sample (blank) quantification value was subtracted from the quantification value of the biotin-added plasma sample obtained in Table 7, and the accuracy (% RE), accuracy (% CV), and recovery rate (%) were calculated as above. The results calculated by equations (1) to (3) are shown. In the sample added with 0.5 ng / mL, the accuracy (% CV) is 2.2% and the accuracy (% RE) is −3.4%, and the judgment criteria (% CV: 15.0% or less,% RE: Within ± 15.0%).
実施例３
健常人および透析患者の血漿中ビオチン濃度を固定相がオクタデシルシリル（ＯＤＳ）（Ｃ１８）であるカラム（ＡＣＱＵＩＴＹ ＵＰＬＣ ＨＳＳ Ｔ３、ウォーターズ製）を用いた分析法（以下、「ＯＤＳ法」）、ペンタフルオロフェニル(ＰＦＰ)カラム（Ａｓｃｅｎｔｉｓ Ｅｘｐｒｅｓｓ Ｆ５ ＨＰＬＣ ｃｏｌｕｍｎ、シグマ-アルドリッチ製）を用いた分析法（以下、「ＰＦＰ法」）により測定した。
Example 3
Analysis method of biotin concentration in healthy and dialysis patients using column (ACQUITY UPLC HSS T3, manufactured by Waters) whose stationary phase is octadecylsilyl (ODS) (C18) (hereinafter referred to as “ODS method”), pentafluoro It measured by the analysis method (henceforth "PFP method") using the phenyl (PFP) column (Ascentis Express F5 HPLC column, the product made from Sigma-Aldrich).
１. 実験方法
血漿試料は、健常人および透析患者より採血し、分離した血漿３００μＬにメタノール３０μＬを添加したものを使用した。
検量線用試料の調製は、ＯＤＳ法、ＰＦＰ法共に実施例２の条件にて行った。また、分析方法は、ＯＤＳ法は実施例１、ＰＦＰ法は実施例２の条件にしたがって測定した。
試料前処理法については、検量線用試料、ゼロ試料および血漿試料には内標準溶液（ＩＳＳ-３）を３０μＬ添加し、ブランク試料にはメタノールを３０μＬ添加した。その後は実施例２の３．（２）〜（５）の手順に従い行った。
1. Experimental Method As a plasma sample, a sample obtained by collecting blood from a healthy person and a dialysis patient and adding 30 μL of methanol to 300 μL of separated plasma was used.
The calibration curve sample was prepared under the conditions of Example 2 for both the ODS method and the PFP method. Moreover, the analysis method measured according to the conditions of Example 1 for the ODS method and Example 2 for the PFP method.
Regarding the sample pretreatment method, 30 μL of the internal standard solution (ISS-3) was added to the calibration curve sample, zero sample, and plasma sample, and 30 μL of methanol was added to the blank sample. After that, the second example 3. This was performed according to the procedures (2) to (5).
２. 結果
２．１. ビオチンピークの分離
図７に示されるように、ＯＤＳ法では、ＭＲＭクロマトグラム上でビオチンのピークに夾雑ピークが重なりピークの分離が困難であった。特に透析患者の血漿においてこの傾向が強く見られた。一方、ＰＦＰ法を用いた場合、ビオチンのピークを分離することができた。
２．２. イオン化抑制
表８に示されるように、ＯＤＳ法では、マトリックス効果によるイオン化抑制が強く、内標準物質で比較すると、透析患者の血漿において、標準液のピーク面積に対して５０％以下の面積値となる血漿試料が散見されたが、ＰＦＰ法ではイオン化抑制が改善された。これにより、低濃度の血漿中ビオチンピークを検出することができた。
２．３. 定量限界値
上記のように、ＰＦＰ法は、ＯＤＳ法に比べてビオチンピークの分離およびイオン化抑制の点で改善されていた。これにより、ビオチンの定量限界値は、ＯＤＳ法が０．１ｎｇ／ｍＬであるのに対して、ＰＦＰ法は０．０５ｎｇ／ｍＬであり、高感度であった。
2. Results 2.1. Separation of Biotin Peaks As shown in FIG. 7, in the ODS method, it was difficult to separate peaks by overlapping a biotin peak on the MRM chromatogram. This tendency was particularly strong in the plasma of dialysis patients. On the other hand, when the PFP method was used, the biotin peak could be separated.
2.2. Suppression of ionization As shown in Table 8, in the ODS method, the suppression of ionization due to the matrix effect is strong. Compared with the internal standard substance, the plasma of dialysis patients has 50% or less of the peak area of the standard solution. Although some plasma samples with an area value of 2 were observed, ionization suppression was improved by the PFP method. Thereby, a biotin peak in plasma at a low concentration could be detected.
2.3. Limit of Quantification As described above, the PFP method was improved in terms of separation of biotin peaks and suppression of ionization compared to the ODS method. Thereby, the quantification limit value of biotin was 0.1 ng / mL in the ODS method, and 0.05 ng / mL in the PFP method, which was highly sensitive.
実施例４
血液透析患者の血液透析（ＨＤ）廃液中ビオチン濃度をＰＦＰ法により測定した。
１. 実験方法
血液透析廃液試料は、血液透析患者の血液透析廃液（ＨＤ廃液１〜４）３００μＬにメタノール３０μＬを添加したものを使用した。
検量線用試料の調製および分析方法は、実施例２の条件にて行った。また、試料前処理法については、検量線試料およびビオチン測定用血液透析廃液試料には内標準物質（ＩＳＳ-４）３０μＬを、妨害ピーク確認用血液透析廃液試料についてはメタノール３０μＬを添加し、以後は実施例２の３．（２）〜（５）に従い行った。
２. 結果
２．１. 血液透析廃液試料中妨害ピークの確認
図８に血液透析廃液試料および検量線試料（０．５ｎｇ／ｍＬ）のクロマトグラムを示した。また、内標準物質であるｒａｃビオチン-ｄ４について、血液透析廃液中に妨害と成り得るピークは確認できなかった（図８Ｄ）。
２．２. 血液透析廃液試料中ビオチン濃度
表９に血液透析廃液中ビオチン濃度の測定結果を示した。
Example 4
The biotin concentration in the hemodialysis (HD) waste solution of hemodialysis patients was measured by the PFP method.
1. Experimental Method As a hemodialysis waste liquid sample, 300 μL of hemodialysis waste liquid (
The calibration curve sample was prepared and analyzed under the conditions of Example 2. For the sample pretreatment method, 30 μL of internal standard substance (ISS-4) was added to the calibration curve sample and the hemodialysis waste liquid sample for biotin measurement, and 30 μL of methanol was added to the hemodialysis waste liquid sample for checking the interference peak. 2 in Example 2. (2) to (5).
2. Results 2.1. Confirmation of Interference Peak in Hemodialysis Waste Solution Sample FIG. 8 shows chromatograms of the hemodialysis waste solution sample and the calibration curve sample (0.5 ng / mL). In addition, for rac biotin-d4, which is an internal standard substance, no peak that could interfere with the hemodialysis waste liquid could be confirmed (FIG. 8D).
2.2. Biotin concentration in hemodialysis waste liquid sample Table 9 shows the measurement results of biotin concentration in hemodialysis waste liquid.
実施例５
血液透析濾過（ＨＤＦ）廃液中のビオチン濃度をＰＦＰ法により測定した。
１. 実験方法
血液透析濾過試料は血液透析濾過患者の廃液（ＨＤＦ廃液）３００μＬにメタノール３０μＬを添加したものを使用した。
検量線用試料の調整および分析方法については、実施例２の条件にて行った。また、試料前処理法については実施例４に従って行った。
２. 結果
２．１. 血液透析濾過試料中妨害ピークの確認
図９に血液透析濾過廃液試料および検量線試料（０．５ｎｇ／ｍＬ）のクロマトグラムを示した。内標準物質であるｒａｃビオチン-ｄ４について、血液透析濾過廃液中に妨害と成り得るピークは確認できなかった（図９Ｄ）。
２．２. 血液透析濾過廃液試料中ビオチン濃度
表１０に血液透析濾過廃液中ビオチン濃度の測定結果を示した。
Example 5
The biotin concentration in the hemodiafiltration (HDF) waste solution was measured by the PFP method.
1. Experimental method The hemodiafiltration sample used was obtained by adding 30 μL of methanol to 300 μL of a hemodiafiltration patient waste liquid (HDF waste liquid).
The calibration curve sample preparation and analysis method were performed under the conditions of Example 2. The sample pretreatment method was performed according to Example 4.
2. Result 2.1. Confirmation of interference peak in hemodiafiltration sample FIG. 9 shows chromatograms of the hemodiafiltration waste solution sample and the calibration curve sample (0.5 ng / mL). For rac biotin-d4, which is an internal standard substance, no peak that could interfere with hemodiafiltration was not confirmed (FIG. 9D).
2.2. Biotin concentration in hemodiafiltration waste liquid sample Table 10 shows the measurement results of biotin concentration in hemodiafiltration waste liquid.
実施例６
腹膜透析廃液中のビオチン濃度をＰＦＰ法により測定した。
１. 実験方法
腹膜透析廃液試料は、腹膜透析患者の廃液（ＰＤ廃液）３００μＬにメタノール３０μＬを添加したものを使用した。
検量線用試料の調整および分析方法については、実施例２の条件にて行った。試料前処理法は、実施例４の条件にて行った。
２. 結果
２．１. 腹膜透析廃液試料中妨害ピークの確認
図１０に腹膜透析廃液試料および検量線試料（２.０ｎｇ／ｍＬ）のクロマトグラムを示した。内標準物質であるｒａｃビオチン-ｄ４について、腹膜透析廃液中に妨害と成り得るピークは確認できなかった（図１０Ｄ）。
２．２. 腹膜透析廃液試料中ビオチン濃度
表１１に腹膜透析廃液中ビオチン濃度の測定結果を示した。
Example 6
The biotin concentration in the peritoneal dialysis waste liquid was measured by the PFP method.
1. Experimental Method The peritoneal dialysis waste liquid sample used was a peritoneal dialysis patient waste liquid (PD waste liquid) 300 μL added with 30 μL of methanol.
The calibration curve sample preparation and analysis method were performed under the conditions of Example 2. The sample pretreatment method was performed under the conditions of Example 4.
2. Results 2.1. Confirmation of Interference Peak in Peritoneal Dialysis Waste Solution Sample FIG. 10 shows chromatograms of the peritoneal dialysis waste solution sample and the calibration curve sample (2.0 ng / mL). For rac biotin-d4, which is an internal standard substance, no peak that could interfere with peritoneal dialysis waste liquid could be confirmed (FIG. 10D).
2.2. Biotin concentration in peritoneal dialysis waste liquid sample Table 11 shows the measurement results of biotin concentration in peritoneal dialysis waste liquid.
実施例７
尿中のビオチン濃度をＰＦＰ法により測定した。
１. 実験方法
検量線用試料の調整および分析方法については、実施例２の条件にて行った。尿試料は、血液透析患者の尿（尿１：透析治療歴約１年の糖尿病患者、尿２：透析治療歴約３年の非糖尿病患者）および蛋白尿（尿３：尿蛋白２＋）各３００μＬにメタノール３０μＬを添加したものを使用した。試料前処理法は、実施例４の条件にて行った。
２. 結果
２．１. 透析廃液試料中妨害ピークの確認
図１１に尿試料および検量線試料（２.０ｎｇ／ｍＬ）のクロマトグラムを示した。内標準物質であるｒａｃビオチン-ｄ４について、尿中に妨害と成り得るピークは確認できなかった（図１１Ｄ）。
２．２. 尿試料中ビオチン濃度
表１２に尿中ビオチン濃度の測定結果を示した。
Example 7
The biotin concentration in urine was measured by the PFP method.
1. Experimental Method The calibration curve sample was prepared and analyzed under the conditions of Example 2. The urine samples were 300 μL each of urine from hemodialysis patients (urine 1: diabetic patient with about 1 year of dialysis treatment, urine 2: non-diabetic patient with about 3 years of dialysis treatment) and proteinuria (urine 3:
2. Results 2.1. Confirmation of interference peak in dialysis waste liquid sample FIG. 11 shows chromatograms of a urine sample and a calibration curve sample (2.0 ng / mL). For rac biotin-d4, which is an internal standard substance, no peak that could interfere with urine could be confirmed (FIG. 11D).
2.2. Biotin concentration in urine sample Table 12 shows the measurement results of biotin concentration in urine.
Claims (14)
（１）メタノールを用いて生体試料中のタンパク質を沈殿させることにより除去する工程と、
（２）工程（１）で得られた試料中のビオチンまたはビオチン関連物質を、高速液体クロマトグラフ／タンデム質量分析法を用いて測定する工程と
を含む、方法。 A method for measuring biotin or a biotin-related substance in a biological sample,
(1) removing the protein in the biological sample by precipitation using methanol ;
(2) measuring the biotin or biotin-related substance in the sample obtained in step (1) using high performance liquid chromatography / tandem mass spectrometry.
のいずれかに記載の方法。 Column used in the high performance liquid chromatograph, hydrophobic interactions, dipole - a column for separating the dipole interaction, and [pi-[pi interaction, claim 1-11
The method in any one of.
（１）生体試料中のタンパク質を除去する工程に使用するための、メタノールを含む試薬と、
（２）高速液体クロマトグラフ／タンデム質量分析法に使用するための機器
を含む、キット。 A measurement kit for measuring biotin or a biotin-related substance in a biological sample,
(1) a reagent containing methanol for use in the step of removing proteins in a biological sample;
(2) A kit comprising equipment for use in high performance liquid chromatograph / tandem mass spectrometry.
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