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Determination of plasma heparin by micellar electrokinetic capillary chromatography1
Talanta 46 (1998) 757 – 760
Short communication
Determination of plasma heparin by micellar electrokinetic capillary chromatography1 Xiao-Mian Zhou *, Jian-Wu Liu, Meng-En Zhang, Shun-jin Chen Department of Medical Laboratory, NanFang Hospital, Guangzhou 510515, People’s Republic of China Received 19 February 1997; received in revised form 14 August 1997; accepted 18 August 1997
Abstract The micellar electrokinetic capillary chromatography (MECC) method is reported for the separation of heparin, and for the possibility of direct determination of free heparin in plasma. The conditions for MECC were: pH 8.5, 25 mM sodium dodecyl sulfate (SDS), 25 mM borate buffer, with a 30 cm × 50 mm ID fused-silica capillary. The sample was detected with a UV-detector at 270 nm with heparin as external standard. The recovery rate was 95.6 – 98.7%. This method was linear in the range 80–7000 U l − 1. The within-run and between-run relative standard deviations were lower than 3.1 and 4.5%, respectively. It is suggested that this MECC method may be used to determine blood samples containing high levels of heparin. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Heparin; High-performance capillary electrophoresis; Plasma
1. Introduction Blood coagulation tests are used mostly to monitor plasma heparin levels in clinical therapy: tests such as the thrombin time (TT), activated partial thrombokinase time (APTT), etc., which measure anticoagulation activity of heparin in blood. However, these methods cannot quantitate the concentration of plasma heparin. Several tech* Corresponding author. 1 Presented at the First Asia-Pacific International Symposium on Capillary Electrophoresis, and other Nano- and Microscale Analytical Techniques, held in Singapore, December 17 – 20, 1996.
niques have been investigated for the analysis of heparin preparations. Gradient polyacrylamide gel electrophoresis (PAGE) [1] and strong anion exchange high-performance liquid chromatography (HPLC) [2] have been the methods of choice for the qualitative and quantitative analysis of heparin preparations. Recently, a number of workers who carried out investigations [3–5] reported that high-performance capillary electrophoresis (HPCE) was a sensitive, highresolution method for the determination of the heparin fragment. However, the samples described above in the papers were non-biological samples. Mao et al. [6] reported that free heparin
0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0039-9140(98)00040-X
758
X.-M. Zhou et al. / Talanta 46 (1998) 757–760
in plasma was separated and its concentration was successfully determined by HPLC. HPLC is, however, a time-consuming, labor-intensive and high-cost method. This paper describes and compares different methods for determining plasma heparin. The MECC method is simple, rapid, highly sensitive, especially suitable for determining a high dosage of heparin, and not affected by fibrin decomposing product (FDP) and other factors.
2. Material and methods
2.1. Instrument The instrument used in this work is the automated BioFocus 3000 capillary electrophoresis system (Bio-Rad, USA) with a diode array UV detector, an automatic injector, and a fluid cooled cartridge. The capillary cassette used was filled with a 50 mm ID fused-silica column, 30 cm in length (to detector). Injection of the sample was by pressure for 8 s. All electrophoresis was carried out at 20°C, with an applied voltage of 20 kV and UV detection at 270 nm. The electrophoresis buffer was boric acid (25 mmol l − 1), sodium dodecyl sulfate (SDS) (25 mmol l − 1), adjusted to pH 8.5 with 1 N sodium hydroxide. Sodium heparin solution (250000 u l − 1) was purchased from Shangshia Biochemistry Pharmaceutical Factory. The anticoagulation activity of sodium heparin was calibrated with the standard solution of sodium heparin (355000 u l − 1, US Pharmacopeia). The sodium heparin accorded with the unit marked by the factory.
2.2. Methods Object of study: 10 extraneous circulation patients (360– 500 units sodium heparin per kg body weight), 15 dialysis patients (10 patients, 65 u kg − 1; five patients, 10.5 u kg − 1). Preparation of samples: after 30 min of intravenous injection, 2 ml blood was collected. Plasma was isolated by centrifugation at 3000×
g for 10 min. A total of 200 ul plasma was deproteinized by mixing with 300 ul acetonitrile for 2 min and then centrifuging for 5 min at 3000× g. Heparin plasma may be stored at − 20°C for 1 week, but cannot be subjected repeatedly to a freeze/thaw cycle. Stock standard solution: stock standard solution was prepared by diluting 250000 u l − 1 sodium heparin 50-fold with redistilled water. Working standard solutions: the stock standard solution was diluted to concentration levels that bracketed the concentration level of interest, prior to analysis. Electrophoresis: the supernatants from the acetonitrile deproteinization were introduced into the capillary by pressure injection. After each sample, the capillary was washed with water (1 min); 1 N NaOH (1 min); and fresh buffer by pressure injection. The capillary was washed daily with NaOH, 1 N (2 min); water (1 min); and finally, electrophoresis buffer.
3. Results Sodium heparin has maximum absorbance at 270 nm. Fig. 1 illustrates the peak of sodium heparin, which is single, intact, symmetrical, with good separation, its migration time being 2.589 0.02 min. The working standard solution was diluted to 0.08, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 u ml − 1. The peak area and the amount of heparin were linear in the range 80–7000 u l − 1 with the detection limit of 25 u l − 1 (y= 131253.1x −13895.6, r=0.998). A known amount of heparin was added to 1 ml plasma of a normal person in the manner described above, and the peak area and the standard peak area were measured. Recovery rates of 95.6–98.7% were obtained. With regard to the reproducibility of different concentrations of heparin, the within-run R.S.D. and between-run R.S.D. were 1.5–3.0 and 2.0–4.4%, respectively. For comparison, the results of the improved TT [7] and HPCE were similar (P\0.05) (see Table 1).
X.-M. Zhou et al. / Talanta 46 (1998) 757–760
759
Fig. 1. (A) Electrophoretograms of blank plasma. (B) Electrophoretograms of heparin plasma: (a) sodium heparin; (b) protein peak. Conditions: pH 8.5; 25 mmol l − 1 SDS; 25 mmol l − 1 borate buffer.
4. Discussion Our experimental results show good separation of heparin by HPCE. The peak is single, intact and symmetrical. The peak area and the concentration of plasma heparin are strongly linear. HPCE may quantitate free heparin in plasma. During deproteinization, deproteinization with acetonitrile was greater than with methanol, ethanol, trichloroacetic acid, or methanol combined with acetone. Acetonitrile deproteinization allows a larger sample volume to be introduced into the capillary and increases the plate number and peak height [8], thereby increasing the sensitivity of the material determined. When heparin was injected into the blood circulation, its anticoagulation activity depended on the amount of heparin in blood. Our results
Table 1 Comparison of the results from the two methods Group
Extraneous circulation Blood dialysis Dose of 65 u kg−1 Dose of 10.5 u kg−1
n
Conc. of plasma heparin (u l−1, x9 s) TT
HPCE
10
5100 9 510
52309490
10 5
2909 100 1009 20
3009110 110930
show that free heparin in plasma is basically in direct proportion to the dose of heparin. The concentration of free heparin measured by HPCE was similar to the activity of plasma heparin determined by the improved TT. Hence, the concentration of plasma heparin determined by HPCE may stand for the activity of heparin anticoagulation. It is essential to monitor high doses of heparin in plasma in clinical therapy. The HPCE method may determine higher doses of heparin: the results of three patients were measured at 5958, 6270 and 5876 u l − 1, respectively. However, the clotting tests were difficult to use to measure them and their reproducibility was very poor. When the concentrations of FDP of two patients were 75 and 96 mg l − 1, the concentrations of free heparin by HPCE were 152 and 276 u l − 1, respectively. However, it is very difficult to measure the levels of heparin by the improved TT method. HPCE has the advantage that it is not affected by the level of fibrinogen, FDP and antithrombin-III, and the operation is simple to perform.
5. Conclusion The HPCE method plasma heparin and measuring high doses apy. Its operation is sensitive.
may be used to quantitate is especially suitable for of heparin in clinical thersimple, rapid, and highly
760
X.-M. Zhou et al. / Talanta 46 (1998) 757–760
References [1] R.E. Edens, A. Al-Hakim, J.W. Weiler, et al., J. Pharm. Sci. 81 (1992) 823–827. [2] R.J. Linhardt, K.G. Rice, Y.S. Kim, et al., Biochem. J. 254 (1988) 781–787. [3] U.K. Desai, H.M. Wang, S.A. Ampofo, et al., Anal. Biochem. 213 (1993) 120–127.
.
[4] J.B.L. Damm, G.J. Overkolift, J. Chromatogr. A 678 (1994) 151 – 165. [5] K. Malsch, J. Harenberg, D.L. Heene, J. Chromatogr A 716 (1995) 259. [6] P. Mao, S. Huang, C. Li, et al., Chin. J. Med. Lab. Technol. 18 (6) (1995) 358 – 360. [7] Z.K. Shihabi, J. Chromatogr. A 652 (1993) 471 – 475. [8] P. Mao, F. Chen, C. Li, Chin. J. Med. Lab. Technol. 18 (5) (1995) 295.
Short communication
Determination of plasma heparin by micellar electrokinetic capillary chromatography1 Xiao-Mian Zhou *, Jian-Wu Liu, Meng-En Zhang, Shun-jin Chen Department of Medical Laboratory, NanFang Hospital, Guangzhou 510515, People’s Republic of China Received 19 February 1997; received in revised form 14 August 1997; accepted 18 August 1997
Abstract The micellar electrokinetic capillary chromatography (MECC) method is reported for the separation of heparin, and for the possibility of direct determination of free heparin in plasma. The conditions for MECC were: pH 8.5, 25 mM sodium dodecyl sulfate (SDS), 25 mM borate buffer, with a 30 cm × 50 mm ID fused-silica capillary. The sample was detected with a UV-detector at 270 nm with heparin as external standard. The recovery rate was 95.6 – 98.7%. This method was linear in the range 80–7000 U l − 1. The within-run and between-run relative standard deviations were lower than 3.1 and 4.5%, respectively. It is suggested that this MECC method may be used to determine blood samples containing high levels of heparin. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Heparin; High-performance capillary electrophoresis; Plasma
1. Introduction Blood coagulation tests are used mostly to monitor plasma heparin levels in clinical therapy: tests such as the thrombin time (TT), activated partial thrombokinase time (APTT), etc., which measure anticoagulation activity of heparin in blood. However, these methods cannot quantitate the concentration of plasma heparin. Several tech* Corresponding author. 1 Presented at the First Asia-Pacific International Symposium on Capillary Electrophoresis, and other Nano- and Microscale Analytical Techniques, held in Singapore, December 17 – 20, 1996.
niques have been investigated for the analysis of heparin preparations. Gradient polyacrylamide gel electrophoresis (PAGE) [1] and strong anion exchange high-performance liquid chromatography (HPLC) [2] have been the methods of choice for the qualitative and quantitative analysis of heparin preparations. Recently, a number of workers who carried out investigations [3–5] reported that high-performance capillary electrophoresis (HPCE) was a sensitive, highresolution method for the determination of the heparin fragment. However, the samples described above in the papers were non-biological samples. Mao et al. [6] reported that free heparin
0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0039-9140(98)00040-X
758
X.-M. Zhou et al. / Talanta 46 (1998) 757–760
in plasma was separated and its concentration was successfully determined by HPLC. HPLC is, however, a time-consuming, labor-intensive and high-cost method. This paper describes and compares different methods for determining plasma heparin. The MECC method is simple, rapid, highly sensitive, especially suitable for determining a high dosage of heparin, and not affected by fibrin decomposing product (FDP) and other factors.
2. Material and methods
2.1. Instrument The instrument used in this work is the automated BioFocus 3000 capillary electrophoresis system (Bio-Rad, USA) with a diode array UV detector, an automatic injector, and a fluid cooled cartridge. The capillary cassette used was filled with a 50 mm ID fused-silica column, 30 cm in length (to detector). Injection of the sample was by pressure for 8 s. All electrophoresis was carried out at 20°C, with an applied voltage of 20 kV and UV detection at 270 nm. The electrophoresis buffer was boric acid (25 mmol l − 1), sodium dodecyl sulfate (SDS) (25 mmol l − 1), adjusted to pH 8.5 with 1 N sodium hydroxide. Sodium heparin solution (250000 u l − 1) was purchased from Shangshia Biochemistry Pharmaceutical Factory. The anticoagulation activity of sodium heparin was calibrated with the standard solution of sodium heparin (355000 u l − 1, US Pharmacopeia). The sodium heparin accorded with the unit marked by the factory.
2.2. Methods Object of study: 10 extraneous circulation patients (360– 500 units sodium heparin per kg body weight), 15 dialysis patients (10 patients, 65 u kg − 1; five patients, 10.5 u kg − 1). Preparation of samples: after 30 min of intravenous injection, 2 ml blood was collected. Plasma was isolated by centrifugation at 3000×
g for 10 min. A total of 200 ul plasma was deproteinized by mixing with 300 ul acetonitrile for 2 min and then centrifuging for 5 min at 3000× g. Heparin plasma may be stored at − 20°C for 1 week, but cannot be subjected repeatedly to a freeze/thaw cycle. Stock standard solution: stock standard solution was prepared by diluting 250000 u l − 1 sodium heparin 50-fold with redistilled water. Working standard solutions: the stock standard solution was diluted to concentration levels that bracketed the concentration level of interest, prior to analysis. Electrophoresis: the supernatants from the acetonitrile deproteinization were introduced into the capillary by pressure injection. After each sample, the capillary was washed with water (1 min); 1 N NaOH (1 min); and fresh buffer by pressure injection. The capillary was washed daily with NaOH, 1 N (2 min); water (1 min); and finally, electrophoresis buffer.
3. Results Sodium heparin has maximum absorbance at 270 nm. Fig. 1 illustrates the peak of sodium heparin, which is single, intact, symmetrical, with good separation, its migration time being 2.589 0.02 min. The working standard solution was diluted to 0.08, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 u ml − 1. The peak area and the amount of heparin were linear in the range 80–7000 u l − 1 with the detection limit of 25 u l − 1 (y= 131253.1x −13895.6, r=0.998). A known amount of heparin was added to 1 ml plasma of a normal person in the manner described above, and the peak area and the standard peak area were measured. Recovery rates of 95.6–98.7% were obtained. With regard to the reproducibility of different concentrations of heparin, the within-run R.S.D. and between-run R.S.D. were 1.5–3.0 and 2.0–4.4%, respectively. For comparison, the results of the improved TT [7] and HPCE were similar (P\0.05) (see Table 1).
X.-M. Zhou et al. / Talanta 46 (1998) 757–760
759
Fig. 1. (A) Electrophoretograms of blank plasma. (B) Electrophoretograms of heparin plasma: (a) sodium heparin; (b) protein peak. Conditions: pH 8.5; 25 mmol l − 1 SDS; 25 mmol l − 1 borate buffer.
4. Discussion Our experimental results show good separation of heparin by HPCE. The peak is single, intact and symmetrical. The peak area and the concentration of plasma heparin are strongly linear. HPCE may quantitate free heparin in plasma. During deproteinization, deproteinization with acetonitrile was greater than with methanol, ethanol, trichloroacetic acid, or methanol combined with acetone. Acetonitrile deproteinization allows a larger sample volume to be introduced into the capillary and increases the plate number and peak height [8], thereby increasing the sensitivity of the material determined. When heparin was injected into the blood circulation, its anticoagulation activity depended on the amount of heparin in blood. Our results
Table 1 Comparison of the results from the two methods Group
Extraneous circulation Blood dialysis Dose of 65 u kg−1 Dose of 10.5 u kg−1
n
Conc. of plasma heparin (u l−1, x9 s) TT
HPCE
10
5100 9 510
52309490
10 5
2909 100 1009 20
3009110 110930
show that free heparin in plasma is basically in direct proportion to the dose of heparin. The concentration of free heparin measured by HPCE was similar to the activity of plasma heparin determined by the improved TT. Hence, the concentration of plasma heparin determined by HPCE may stand for the activity of heparin anticoagulation. It is essential to monitor high doses of heparin in plasma in clinical therapy. The HPCE method may determine higher doses of heparin: the results of three patients were measured at 5958, 6270 and 5876 u l − 1, respectively. However, the clotting tests were difficult to use to measure them and their reproducibility was very poor. When the concentrations of FDP of two patients were 75 and 96 mg l − 1, the concentrations of free heparin by HPCE were 152 and 276 u l − 1, respectively. However, it is very difficult to measure the levels of heparin by the improved TT method. HPCE has the advantage that it is not affected by the level of fibrinogen, FDP and antithrombin-III, and the operation is simple to perform.
5. Conclusion The HPCE method plasma heparin and measuring high doses apy. Its operation is sensitive.
may be used to quantitate is especially suitable for of heparin in clinical thersimple, rapid, and highly
760
X.-M. Zhou et al. / Talanta 46 (1998) 757–760
References [1] R.E. Edens, A. Al-Hakim, J.W. Weiler, et al., J. Pharm. Sci. 81 (1992) 823–827. [2] R.J. Linhardt, K.G. Rice, Y.S. Kim, et al., Biochem. J. 254 (1988) 781–787. [3] U.K. Desai, H.M. Wang, S.A. Ampofo, et al., Anal. Biochem. 213 (1993) 120–127.
.
[4] J.B.L. Damm, G.J. Overkolift, J. Chromatogr. A 678 (1994) 151 – 165. [5] K. Malsch, J. Harenberg, D.L. Heene, J. Chromatogr A 716 (1995) 259. [6] P. Mao, S. Huang, C. Li, et al., Chin. J. Med. Lab. Technol. 18 (6) (1995) 358 – 360. [7] Z.K. Shihabi, J. Chromatogr. A 652 (1993) 471 – 475. [8] P. Mao, F. Chen, C. Li, Chin. J. Med. Lab. Technol. 18 (5) (1995) 295.