反相-高效液相色谱法分析血清多胺含量
作者：吴逸明 张振中 史香林 吴如金
单位：吴逸明(河南医科大学劳动卫生学与卫生毒理学教研室 郑州 450052)；张振中(河南医科大学药学系 郑州 450052)；史香林(美国职业安全卫生研究所病理生理学研究室 摩根墩 WV26505 USA)；吴如金(中国药科大学药物分析教研室 南京 210009)
关键词：多胺；血清；RP-HPLC；方法
河南医科大学学报000313 摘要 目的：建立血清多胺反相-高效液相色谱(RP_HPLC)分析方法。方法: 以邻苯二甲醛(OPA) 作为荧光衍生试剂, 采用ODS-C18柱(200 mm × 4.6 mm I.D , 2μm); 柱温, 35℃; 采用梯度洗脱(A液, V(水)∶V(磷酸)∶ V(三乙胺)= 360∶9∶6; B液, 甲醇); 流速, 0.8 ml/min; 检测波长, 338 nm。结果: 建立了反相-高效液相色谱测定血清多胺的分析方法。结论: 该方法可用于大鼠血清等生物样品多胺的测定。
, 百拇医药
分类号 R313
Determination of polyamines in serum by RP-HPLC
WU Yiming ,(Department of Occupational Health and Health Toxicology, Henan Medical University， Zhengzhou 450052)
ZHANG Zhenzhong,(Department of Pharmacy, Henan Medical University， Zhengzhou 450052)
SHI Xianglin，(Pathology and Physiology Research, NIOSH, Morgantown, WV 26505, USA)
, 百拇医药
WU Rujin
(Department of Pharmaceutical Analysis,China Pharmaceutical University,Nanjing 210009)
Abstract Aim: To establish determination method of polyamines (RP-HPLC) in serum. Methods: O-phenyldehyde (OPA) was used as derivative reagent . Chromatography conditions were as the following: ODS-C18 column (200 mm × 4.6 mm I.D ,2μm); column temperature , 35 ℃ ; gradient elution (A， V(water)∶ V (phosphoric acid)∶V (triethylamine)= 360∶9∶6; B, methanol) ; flow rate , 0.8 ml/min ; detection wavelength , 338 nm. Results and Conclusion: The method with the characteristics of rapidity and accuracy is suitable for the determination of polyamines in serum.
, 百拇医药
Key words polyamines; serum; RP-HPLC;methods
Polyamines which are low-molecular-weight fatty family compounds and contain multiamino and (or) sub-amino groups, present in all living cells widely. Polyamines mainly include putrescine, spermidine, spermine and their derivatives. They have been implicated in cell growth, proliferation and differentiation, and affect the metabolism of DNA, RNA and proteins. Many studies showed that polyamines levels were associated with tumor development and progression[1,2]. In recent literatures[3,4], polyamines were mainly determined by HPLC. However, these methods had the disadvantages of long time of analysis and couldn′t be used easily in practical samples. In this study, a rapid method of RP-HPLC was established and used to determine polyamines in serum.
, 百拇医药
1 Materials and methods
1.1 Apparatus Waters HPLC system (including Waters 600 pump, Waters 486 detector whose wavelength can be changed, Waters 800 integration), high-speed centrifugal machine (Shanghai Medical Centrifugal Factory), Millipore pure water system(American Millipore Company)
1.2 Reagents spermidine hydrochloride (Sigma Company), spermine hydrochloride (Sigma Company), putrescine hydrochloride (Sigma Company), methanol (A.R)( Tianjin Reagents Factory), mercaptoalcohol(A.R)( Shanghai No. 4 Reagents Factory), triethylamine(Shanghai No. 3 Reagents Factory), phenyldehyde(OPA)(C.P)(Tongji University), perchloric acid (A.R)(Beijing Hongxing Chemical Factory ), potassium hydroxide (A.R)(Kaifeng Chemical Factory), boric acid (A.R)(Luoyang Chemical Reagents Factory), ethanol (Kaifeng Reagents Factory), chloroform(A.R)(Kaifeng Chemical Reagents Factory), All water used was purified by Millipore pure system.
, http://www.100md.com
1.3 Preparation of reagents
OPA-mercaptoalcohol: adding 50 mg of OPA to 5.0 ml ethanol containing 100 μl of mercaptoalcohol.
0.4 mol/L borate buffer ( pH 10.8): adding 2.4 g boric acid to 25 ml 1.0 mol/L KOH and then adding water to the above solution at final volume of about 100 ml, adjusting pH to 10.8 by use of 1.0 mol/L KOH.
Derivative reagents: adding 9.0 ml of 0.4 mol/L borate buffer to 1.0 ml of OPA-mercaptoalcohol, and mixing well.
, 百拇医药
1.4 Standard stock solution 1.0 mmol/L spermidine, spermine, putrescine and 1,6-hexanediamine were prepared and stored at 4 ℃ until used.
1.5 Pretreatment of samples The samples were pretreated and procedures were as the following: 0.01 ml of internal standard (1,6-hexanediamine) was added to 0.1 ml serum and this solution was diluted with water to a final volume of 0.4 ml. After adding 0.1 ml cool HClO4 (3 mol/L) to the above solution, the mixture was stored at 4 ℃ for 20 min, then centrifuged (11 000 r/min ×10 min).The supernatant was taken, mixed with 0.2 ml phosphate buffer (0.1 mol/L, pH 7.0), 0.1 ml chloroform and 0.06 ml methanol, and centrifuged (11 000 r/min×10 min ). The supernatant was taken and stored at 4 ℃.
, 百拇医药
1.6 Derivatization After blending 20 μl of derivative reagents and 5 μl of samples solution for 90 s , injection was performed immediately.
1.7 Chromatography conditions Chromatography Column, ODS-C18 column (20 mm × 4.6 mm I.D, 2 μm); column temperature, 35 ℃; mobile phase, solution A, V (water)∶V (phosphoric acid)∶V (triethylamine) = 360∶9∶6 ; solution B, methanol, gradient elution(Tab1); flow rate, 0.8 ml/min; detection wavelength, 338 nm. Typical chromatogram was showed in Fig1.
, 百拇医药
Tab1 Gradient elution table t/min
V(A)/%
V(B)/%
0
30
70
3.5
30
70
5.0
20
80
, 百拇医药
6.0
20
80
7.0
0
100
10.0
0
100
11.0
30
70
, 百拇医药
Fig1 Chromatogram of standard polyamines
Spd: spermidine; Spm: spermine; Put: putrescine;
Hex: 1,6-hexanediamine
2 Results
2.1 Standard linear regression equation Standard solutions containing 1,6-hexanediamine (I.S) were injected. In the regression equation , X was the concentration of polyamines and Y was ratio of the peak height of polyamines to that of 1,6-hexanediamine. The linear equations were as the following:
, 百拇医药
Spermine(Spm): Y =-0.72 + 0.065 X ,r = 0.999 3 (1)
Spermidine(Spd): Y = 0.493 + 0.0363 X,r = 0.999 1 (2)
Putrescine(Put): Y = 0.128 + 0.0253 X,r = 0.997 8 (3)
r was the linear regression coefficient.
2.2 The extract linear regression equations of polyamines A series of standard concentration solutions containing 1,6-hexanediamine (I.S) were treated according to sample treatment method, X was the concentration of polyamines and Y was the ratio between the peak height of polyamines and that of 1,6-hexanediamine. the equations were:
, http://www.100md.com
Spermine(Spm): Y = 0.42 + 0.078 X ,r = 0.999 1 (4)
Spermidine(Spd): Y = 0.23 + 0.045 X,r = 0.999 3 (5)
Putrescine(Put): Y = 0.12 + 0.037 X,r =0.999 5 (6)
r was the linear regression coefficient.
2.3 Recovery of extraction After adding polyamines standard solutions to distilled water, the above solutions were treated according to sample treatment method, then adding 1,6-hexanediamine (I.S) , the derivative reaction took place and injection was performed. The recoveries of spermine, spermidine and putrescine were 87.8%, 82.4% and 86.6%, respectively.
, 百拇医药
2.4 Recovery of method Method recovery of spermine, spermidine and putrescine were 102.6%, 98%and 104.2%, respectively.
2.5 Results The established method with the characteristics of rapidity and accuracy was suitable for the determination of polyamines in serum.
基金项目：河南省自然科学基金资助项目 204022300
研究方向：肺癌的病因学、预防、早期诊断和综合治疗，E-mail：ymwu@371.net
作者简介：吴逸明，男，55岁，教授，博士生导师，References
, 百拇医药
[1]Shah N, Thomas T, Shirahata A, Sigal，et al. Activation of nuclear factor kappaB by polyamines in breast cancer cells. Biochemistry,1999，38(45):14 763
[2]Manni A, Wright C, Hsu CJ, et al. Polyamines and autocrine control of tumor growth by prolactin in experimental breast cancer in culture. Endocrinology, 1986, 119: 2 033
[3]Kendra KL, Katzenllenbogen BS. An evaluation of the involvement of polyamines in modulating MCG-7 human breast cancer cell proliferation and progesterone receptor levels by estrogen and antiestrogen . J Steroid Biochem, 1987,28: 123
, 百拇医药
[4]Suh JW, Lee SH, Chung BC, et al. Urinary polyamines evaluation for effective diagnosis of various cancers. J Chromatogra B, 1997, 688:179
[5]Schenkel E, Verlaimont V, Dubois J, et al. Improved HPLC method for the determination of polyamines as their benzoylated derivatives: application to p388 cancer cells. J Chromatogra B,1995,668:189
[6]Huhn G, Mattusch J, Schulz H. Determination of polyamines in biological materials by HPLC with 9-fluorenylmethyl chlorormate precolumn derivatization. Fresenius J Anal Chem, 1995,351:563
2000-01-05收稿, http://www.100md.com
单位：吴逸明(河南医科大学劳动卫生学与卫生毒理学教研室 郑州 450052)；张振中(河南医科大学药学系 郑州 450052)；史香林(美国职业安全卫生研究所病理生理学研究室 摩根墩 WV26505 USA)；吴如金(中国药科大学药物分析教研室 南京 210009)
关键词：多胺；血清；RP-HPLC；方法
河南医科大学学报000313 摘要 目的：建立血清多胺反相-高效液相色谱(RP_HPLC)分析方法。方法: 以邻苯二甲醛(OPA) 作为荧光衍生试剂, 采用ODS-C18柱(200 mm × 4.6 mm I.D , 2μm); 柱温, 35℃; 采用梯度洗脱(A液, V(水)∶V(磷酸)∶ V(三乙胺)= 360∶9∶6; B液, 甲醇); 流速, 0.8 ml/min; 检测波长, 338 nm。结果: 建立了反相-高效液相色谱测定血清多胺的分析方法。结论: 该方法可用于大鼠血清等生物样品多胺的测定。
, 百拇医药
分类号 R313
Determination of polyamines in serum by RP-HPLC
WU Yiming ,(Department of Occupational Health and Health Toxicology, Henan Medical University， Zhengzhou 450052)
ZHANG Zhenzhong,(Department of Pharmacy, Henan Medical University， Zhengzhou 450052)
SHI Xianglin，(Pathology and Physiology Research, NIOSH, Morgantown, WV 26505, USA)
, 百拇医药
WU Rujin
(Department of Pharmaceutical Analysis,China Pharmaceutical University,Nanjing 210009)
Abstract Aim: To establish determination method of polyamines (RP-HPLC) in serum. Methods: O-phenyldehyde (OPA) was used as derivative reagent . Chromatography conditions were as the following: ODS-C18 column (200 mm × 4.6 mm I.D ,2μm); column temperature , 35 ℃ ; gradient elution (A， V(water)∶ V (phosphoric acid)∶V (triethylamine)= 360∶9∶6; B, methanol) ; flow rate , 0.8 ml/min ; detection wavelength , 338 nm. Results and Conclusion: The method with the characteristics of rapidity and accuracy is suitable for the determination of polyamines in serum.
, 百拇医药
Key words polyamines; serum; RP-HPLC;methods
Polyamines which are low-molecular-weight fatty family compounds and contain multiamino and (or) sub-amino groups, present in all living cells widely. Polyamines mainly include putrescine, spermidine, spermine and their derivatives. They have been implicated in cell growth, proliferation and differentiation, and affect the metabolism of DNA, RNA and proteins. Many studies showed that polyamines levels were associated with tumor development and progression[1,2]. In recent literatures[3,4], polyamines were mainly determined by HPLC. However, these methods had the disadvantages of long time of analysis and couldn′t be used easily in practical samples. In this study, a rapid method of RP-HPLC was established and used to determine polyamines in serum.
, 百拇医药
1 Materials and methods
1.1 Apparatus Waters HPLC system (including Waters 600 pump, Waters 486 detector whose wavelength can be changed, Waters 800 integration), high-speed centrifugal machine (Shanghai Medical Centrifugal Factory), Millipore pure water system(American Millipore Company)
1.2 Reagents spermidine hydrochloride (Sigma Company), spermine hydrochloride (Sigma Company), putrescine hydrochloride (Sigma Company), methanol (A.R)( Tianjin Reagents Factory), mercaptoalcohol(A.R)( Shanghai No. 4 Reagents Factory), triethylamine(Shanghai No. 3 Reagents Factory), phenyldehyde(OPA)(C.P)(Tongji University), perchloric acid (A.R)(Beijing Hongxing Chemical Factory ), potassium hydroxide (A.R)(Kaifeng Chemical Factory), boric acid (A.R)(Luoyang Chemical Reagents Factory), ethanol (Kaifeng Reagents Factory), chloroform(A.R)(Kaifeng Chemical Reagents Factory), All water used was purified by Millipore pure system.
, http://www.100md.com
1.3 Preparation of reagents
OPA-mercaptoalcohol: adding 50 mg of OPA to 5.0 ml ethanol containing 100 μl of mercaptoalcohol.
0.4 mol/L borate buffer ( pH 10.8): adding 2.4 g boric acid to 25 ml 1.0 mol/L KOH and then adding water to the above solution at final volume of about 100 ml, adjusting pH to 10.8 by use of 1.0 mol/L KOH.
Derivative reagents: adding 9.0 ml of 0.4 mol/L borate buffer to 1.0 ml of OPA-mercaptoalcohol, and mixing well.
, 百拇医药
1.4 Standard stock solution 1.0 mmol/L spermidine, spermine, putrescine and 1,6-hexanediamine were prepared and stored at 4 ℃ until used.
1.5 Pretreatment of samples The samples were pretreated and procedures were as the following: 0.01 ml of internal standard (1,6-hexanediamine) was added to 0.1 ml serum and this solution was diluted with water to a final volume of 0.4 ml. After adding 0.1 ml cool HClO4 (3 mol/L) to the above solution, the mixture was stored at 4 ℃ for 20 min, then centrifuged (11 000 r/min ×10 min).The supernatant was taken, mixed with 0.2 ml phosphate buffer (0.1 mol/L, pH 7.0), 0.1 ml chloroform and 0.06 ml methanol, and centrifuged (11 000 r/min×10 min ). The supernatant was taken and stored at 4 ℃.
, 百拇医药
1.6 Derivatization After blending 20 μl of derivative reagents and 5 μl of samples solution for 90 s , injection was performed immediately.
1.7 Chromatography conditions Chromatography Column, ODS-C18 column (20 mm × 4.6 mm I.D, 2 μm); column temperature, 35 ℃; mobile phase, solution A, V (water)∶V (phosphoric acid)∶V (triethylamine) = 360∶9∶6 ; solution B, methanol, gradient elution(Tab1); flow rate, 0.8 ml/min; detection wavelength, 338 nm. Typical chromatogram was showed in Fig1.
, 百拇医药
Tab1 Gradient elution table t/min
V(A)/%
V(B)/%
0
30
70
3.5
30
70
5.0
20
80
, 百拇医药
6.0
20
80
7.0
0
100
10.0
0
100
11.0
30
70
, 百拇医药
Fig1 Chromatogram of standard polyamines
Spd: spermidine; Spm: spermine; Put: putrescine;
Hex: 1,6-hexanediamine
2 Results
2.1 Standard linear regression equation Standard solutions containing 1,6-hexanediamine (I.S) were injected. In the regression equation , X was the concentration of polyamines and Y was ratio of the peak height of polyamines to that of 1,6-hexanediamine. The linear equations were as the following:
, 百拇医药
Spermine(Spm): Y =-0.72 + 0.065 X ,r = 0.999 3 (1)
Spermidine(Spd): Y = 0.493 + 0.0363 X,r = 0.999 1 (2)
Putrescine(Put): Y = 0.128 + 0.0253 X,r = 0.997 8 (3)
r was the linear regression coefficient.
2.2 The extract linear regression equations of polyamines A series of standard concentration solutions containing 1,6-hexanediamine (I.S) were treated according to sample treatment method, X was the concentration of polyamines and Y was the ratio between the peak height of polyamines and that of 1,6-hexanediamine. the equations were:
, http://www.100md.com
Spermine(Spm): Y = 0.42 + 0.078 X ,r = 0.999 1 (4)
Spermidine(Spd): Y = 0.23 + 0.045 X,r = 0.999 3 (5)
Putrescine(Put): Y = 0.12 + 0.037 X,r =0.999 5 (6)
r was the linear regression coefficient.
2.3 Recovery of extraction After adding polyamines standard solutions to distilled water, the above solutions were treated according to sample treatment method, then adding 1,6-hexanediamine (I.S) , the derivative reaction took place and injection was performed. The recoveries of spermine, spermidine and putrescine were 87.8%, 82.4% and 86.6%, respectively.
, 百拇医药
2.4 Recovery of method Method recovery of spermine, spermidine and putrescine were 102.6%, 98%and 104.2%, respectively.
2.5 Results The established method with the characteristics of rapidity and accuracy was suitable for the determination of polyamines in serum.
基金项目：河南省自然科学基金资助项目 204022300
研究方向：肺癌的病因学、预防、早期诊断和综合治疗，E-mail：ymwu@371.net
作者简介：吴逸明，男，55岁，教授，博士生导师，References
, 百拇医药
[1]Shah N, Thomas T, Shirahata A, Sigal，et al. Activation of nuclear factor kappaB by polyamines in breast cancer cells. Biochemistry,1999，38(45):14 763
[2]Manni A, Wright C, Hsu CJ, et al. Polyamines and autocrine control of tumor growth by prolactin in experimental breast cancer in culture. Endocrinology, 1986, 119: 2 033
[3]Kendra KL, Katzenllenbogen BS. An evaluation of the involvement of polyamines in modulating MCG-7 human breast cancer cell proliferation and progesterone receptor levels by estrogen and antiestrogen . J Steroid Biochem, 1987,28: 123
, 百拇医药
[4]Suh JW, Lee SH, Chung BC, et al. Urinary polyamines evaluation for effective diagnosis of various cancers. J Chromatogra B, 1997, 688:179
[5]Schenkel E, Verlaimont V, Dubois J, et al. Improved HPLC method for the determination of polyamines as their benzoylated derivatives: application to p388 cancer cells. J Chromatogra B,1995,668:189
[6]Huhn G, Mattusch J, Schulz H. Determination of polyamines in biological materials by HPLC with 9-fluorenylmethyl chlorormate precolumn derivatization. Fresenius J Anal Chem, 1995,351:563
2000-01-05收稿, http://www.100md.com