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Measurement of warfarin in plasma by high performance liquid chromatography (HPLC) and its correlation with the international normalized ratio
Thrombosis Research 111 (2003) 281 – 284
Regular Article
Measurement of warfarin in plasma by high performance liquid chromatography (HPLC) and its correlation with the international normalized ratio Rossana Lombardi, Veena Chantarangkul, Marco Cattaneo, Armando Tripodi * Department of Internal Medicine, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, University and IRCCS Maggiore Hospital, Via Pace 9, Milan 20122, Italy Received 11 September 2003; accepted 11 September 2003
Abstract The measurement of plasma warfarin is required to investigate non-compliance, resistance to anticoagulation, drug metabolism and pharmacokinetic. Methods so far described are based on extraction of warfarin from plasma followed by reversed-phase HPLC. Extraction is the crucial step and may be performed in liquid- or solid-phase. The latter requires the preparation of columns, which makes the procedure variable. We investigated the suitability of the ready-for-use commercial cartridges for sample preparation. The method displayed betweenrun CV of 11.8%. Recovery was 99%. The coefficients of correlation between warfarin concentration in 50 patients and weekly dosage or INR were 0.55 ( p < 0.0001) or 0.25 ( p = 0.079). D 2003 Elsevier Ltd. All rights reserved. Keywords: Warfarin; INR; Anticoagulation; Thrombosis
1. Introduction Vitamin K antagonists are widely used as oral anticoagulants for the treatment of venous thromboembolism as well as for the prevention of systemic embolism in patients with atrial fibrillation and with prosthetic heart valves [1]. The most widely used drugs are warfarin sodium and acenocoumarol. Vitamin K antagonists exert their anticoagulant effect by interfering with the cyclic conversion of vitamin K, thus, inhibiting the carboxylation of glutamate residues to gamma-carboxyglutamate at the N-terminal regions of the vitamin K-dependent coagulation factors (FIX, FVII, FX and FII) and inhibitors (protein C and protein S). The process of gamma-carboxylation is mediated by carboxylase with vitamin K functioning as cofactor. As a result of the antagonizing effect of oral anticoagulants, vitamin K-dependent coagulation factors are synthesized as a-carboxy forms, which do not bind phospholipid surfaces leading to defective coagulation. The dose – response effect of vitamin K antagonists is highly variable and their * Corresponding author. Tel.: +39-2-55035437; fax: +39-2-50320723. E-mail address: [email protected] (A. Tripodi). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2003.09.006
anticoagulant dosage must be closely monitored to prevent over- or under-anticoagulation. The effect of warfarin in patients on oral anticoagulants is usually assessed by the prothrombin time test with results expressed as International Normalized Ratio (INR) [2]. However, the measurement of the plasmatic levels of the drug may be required in some instances to investigate patient compliance, resistance to anticoagulation, drug metabolism and pharmacokinetic. We describe a two-step method whereby warfarin is extracted from plasma with commercially available cartridges followed by quantitation with reversed-phase high performance liquid chromatography (HPLC). We took also the opportunity to investigate patients on oral anticoagulants and to assess the correlation of warfarin concentration vs. the weekly dosage or the INR.
2. Materials and methods 2.1. Cartridges and standard The cartridges were Oasis HLB available from Waters, Milford, MA. They contain a copolymer designed to have a
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2.2. Plasma preparation After informed consent blood was collected from patients on warfarin therapy into vacuum tubes (Vacutainer, Becton and Dickinson, Meylan, France) containing 129 mM trisodium citrate. Blood specimens were centrifuged at 2000 g for 10 min (room temperature) to get platelet-poor plasma, which was snap-frozen in liquid nitrogen and stored at 70 jC until testing. 2.3. Methods Fig. 1. Warfarin calibration curve. AU, absorbance units. Equation describing the relationship, Y = 12568X 60; r2 = 0.999.
hydrophilic – lipophilic balance (HLB) in order to ensure high and reproducible recoveries for acidic, basic and neutral compounds. The standard was pure sodium warfarin (Sigma Aldrich Chemie, Steinheim, Germany) dissolved into distilled water which was then used to spike a pooled normal plasma at (final) concentrations ranging from 0.125 to 5 Ag/ml.
2.3.1. INR determination The INR was measured on fresh plasma by a human recombinant thromboplastin (Instrumentation Laboratory, Orangeburg, NY) combined with an MLA 1600 automated coagulometer (Instrumentation Laboratory). 2.3.2. Warfarin determination The cartridges need conditioning before plasma extraction. This has been performed by methanol followed by equilibration with water. After conditioning, 1 ml of test plasma or standards was applied to the cartridge and the
Fig. 2. Chromatograms obtained with the blank (A) and the standard at 0.125 Ag/ml warfarin concentration (B). AU, absorbance units. The number above the peak identifies warfarin and corresponds to the retention time.
R. Lombardi et al. / Thrombosis Research 111 (2003) 281–284
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the samples containing warfarin at 0.125 Ag/ml and the blank sample are shown in Fig. 2. The between-run reproducibility of the assay, which was estimated as coefficient of variation (CV) by repeat measurements (n = 11) on a plasma sample (warfarin concentration = 1.29 Ag/ml) from a patient on warfarin therapy, was 11.8%. Recovery, estimated by measuring warfarin concentration in five different assays for a drug-free plasma sample pooled from healthy subjects and spiked with warfarin at the final concentration of 2.5 Ag/ml, was 99 F 10.2. The coefficients of correlation between warfarin concentration and the INR or the weekly dosage for 50 treated patients were 0.55 ( p < 0.0001) or 0.25 ( p = 0.079) (Fig. 3).
4. Discussion
Fig. 3. Correlation between plasmatic warfarin concentration and international normalized ratio (INR) (r = 0.25, p = 0.079), or weekly warfarin dosage for 50 patients on oral anticoagulation (r = 0.55, p < 0.001).
unbound material was washed out with 1 ml 5% methanol in water followed by a second washing with 2% acetic acid in 5% methanol in water. The bound material was eluted by 1 ml of 2% ammonium hydroxide in 60% methanol in water. Eluted material was evaporated to dryness under a stream of nitrogen at 40 jC. The residue was eventually dissolved in 100 Al mobile phase and injected (20 Al) into the HPLC system (Waters). Separation was performed on a 3.9 150 mm symmetry shield column (RP8, 5 Am, Waters) and peaks were detected with a tunable absorbance UV detector (Waters) at 280 nm. The eluent (25 mM potassium phosphate, pH 7 with 45% methanol) was applied at a flow rate of 1 ml/min (isocratic conditions). On each test occasion, we run (in duplicate) a set of standards (ranging from 5 to 0.125 Ag/ml) for every seven unknown samples. Peak heights for standards and test plasma were measured and the concentration of warfarin was interpolated from the calibration curve (peak height vs. warfarin concentration).
Methods so far described are based on extraction of warfarin from plasma followed by gas chromatography or reversed-phase HPLC [3 –6]. Extraction is the crucial step and may be performed either in liquid- or solid-phase, which requires careful conditions to ensure good recovery of warfarin before sample injection. The solid-phase extraction requires the preparation of suitable columns, which in addition to the necessary expertise may make the entire assay procedure time-consuming and the sample preparation highly variable. We investigated the suitability of the readyfor-use, commercially available, miniature cartridges for sample preparation in the measurement of warfarin by HPLC. The method is reliable and easy to perform for those laboratories that have access to HPLC equipment. We investigated with this method patients on oral anticoagulants to assess correlation between the concentration of warfarin and the INR or the weekly dosage administered to the patients. Interestingly, the warfarin concentration was more correlated with the weekly dosage than with the INR. This is not surprising. Interaction with other drugs, dependency from diet, seasonal variation and age make the INR poorly dependent on warfarin concentration. This strengthens the concept that warfarin measurement alone would be of little help for dose-adjustment in patients on oral anticoagulants. However, this measurement may be useful in managing patients and should be included in the laboratory armamentarium of oral anticoagulant clinics. In conclusion, the method described proved to be easy to do, reproducible and specific for warfarin. It does not require sophisticated equipment other than the regular HPLC apparatus that is available in clinical laboratories.
3. Results
References
A typical calibration curve (peak height as absorbance unit vs. warfarin concentration) is shown in Fig. 1. Linearity was excellent (r2 = 0.999). Chromatograms correspondent to
[1] Hirsh J, Dalen JE, Anderson DR, Poller L, Bussey H, Ansell J, et al. Oral anticoagulants: mechanism of action, clinical effectiveness, and optimal therapeutic range. Chest 2001;119:8S – 21S. [2] WHO Expert Committee on Biological Standardization. Guidelines for
284
R. Lombardi et al. / Thrombosis Research 111 (2003) 281–284
thromboplastins and plasma used to control oral anticoagulant therapy. Technical Report Series 1999;889:48th report. Geneva, Switzerland. [3] Fasco MJ, Piper LJ, Kaminsky LS. Biochemical applications of a quantitative high-pressure liquid chromatographic assay of warfarin and its metabolites. J Chromatogr 1997;131:365 – 73. [4] Banfield C, Rowland M. Stereospecific fluorescence high-performance liquid chromatographic analysis of warfarin and its metabolites in plasma and urine. J Pharm Sci 1984;73:1392 – 6.
[5] Bush ED, Low LK, Trager WF. A sensitive and specific stable isotope assay for warfarin and its metabolites. Biomed Mass Spectrom 1983;10: 395 – 8. [6] Lang D, Bocker R. Highly sensitive and specific high-performance liquid chromatographic analysis of 7-hydroxywarfarin, a marker for human cytochrome P-4502C9 activity. J Chromatogr B, Biomed Appl 1995;672:305 – 9.
Regular Article
Measurement of warfarin in plasma by high performance liquid chromatography (HPLC) and its correlation with the international normalized ratio Rossana Lombardi, Veena Chantarangkul, Marco Cattaneo, Armando Tripodi * Department of Internal Medicine, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, University and IRCCS Maggiore Hospital, Via Pace 9, Milan 20122, Italy Received 11 September 2003; accepted 11 September 2003
Abstract The measurement of plasma warfarin is required to investigate non-compliance, resistance to anticoagulation, drug metabolism and pharmacokinetic. Methods so far described are based on extraction of warfarin from plasma followed by reversed-phase HPLC. Extraction is the crucial step and may be performed in liquid- or solid-phase. The latter requires the preparation of columns, which makes the procedure variable. We investigated the suitability of the ready-for-use commercial cartridges for sample preparation. The method displayed betweenrun CV of 11.8%. Recovery was 99%. The coefficients of correlation between warfarin concentration in 50 patients and weekly dosage or INR were 0.55 ( p < 0.0001) or 0.25 ( p = 0.079). D 2003 Elsevier Ltd. All rights reserved. Keywords: Warfarin; INR; Anticoagulation; Thrombosis
1. Introduction Vitamin K antagonists are widely used as oral anticoagulants for the treatment of venous thromboembolism as well as for the prevention of systemic embolism in patients with atrial fibrillation and with prosthetic heart valves [1]. The most widely used drugs are warfarin sodium and acenocoumarol. Vitamin K antagonists exert their anticoagulant effect by interfering with the cyclic conversion of vitamin K, thus, inhibiting the carboxylation of glutamate residues to gamma-carboxyglutamate at the N-terminal regions of the vitamin K-dependent coagulation factors (FIX, FVII, FX and FII) and inhibitors (protein C and protein S). The process of gamma-carboxylation is mediated by carboxylase with vitamin K functioning as cofactor. As a result of the antagonizing effect of oral anticoagulants, vitamin K-dependent coagulation factors are synthesized as a-carboxy forms, which do not bind phospholipid surfaces leading to defective coagulation. The dose – response effect of vitamin K antagonists is highly variable and their * Corresponding author. Tel.: +39-2-55035437; fax: +39-2-50320723. E-mail address: [email protected] (A. Tripodi). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2003.09.006
anticoagulant dosage must be closely monitored to prevent over- or under-anticoagulation. The effect of warfarin in patients on oral anticoagulants is usually assessed by the prothrombin time test with results expressed as International Normalized Ratio (INR) [2]. However, the measurement of the plasmatic levels of the drug may be required in some instances to investigate patient compliance, resistance to anticoagulation, drug metabolism and pharmacokinetic. We describe a two-step method whereby warfarin is extracted from plasma with commercially available cartridges followed by quantitation with reversed-phase high performance liquid chromatography (HPLC). We took also the opportunity to investigate patients on oral anticoagulants and to assess the correlation of warfarin concentration vs. the weekly dosage or the INR.
2. Materials and methods 2.1. Cartridges and standard The cartridges were Oasis HLB available from Waters, Milford, MA. They contain a copolymer designed to have a
282
R. Lombardi et al. / Thrombosis Research 111 (2003) 281–284
2.2. Plasma preparation After informed consent blood was collected from patients on warfarin therapy into vacuum tubes (Vacutainer, Becton and Dickinson, Meylan, France) containing 129 mM trisodium citrate. Blood specimens were centrifuged at 2000 g for 10 min (room temperature) to get platelet-poor plasma, which was snap-frozen in liquid nitrogen and stored at 70 jC until testing. 2.3. Methods Fig. 1. Warfarin calibration curve. AU, absorbance units. Equation describing the relationship, Y = 12568X 60; r2 = 0.999.
hydrophilic – lipophilic balance (HLB) in order to ensure high and reproducible recoveries for acidic, basic and neutral compounds. The standard was pure sodium warfarin (Sigma Aldrich Chemie, Steinheim, Germany) dissolved into distilled water which was then used to spike a pooled normal plasma at (final) concentrations ranging from 0.125 to 5 Ag/ml.
2.3.1. INR determination The INR was measured on fresh plasma by a human recombinant thromboplastin (Instrumentation Laboratory, Orangeburg, NY) combined with an MLA 1600 automated coagulometer (Instrumentation Laboratory). 2.3.2. Warfarin determination The cartridges need conditioning before plasma extraction. This has been performed by methanol followed by equilibration with water. After conditioning, 1 ml of test plasma or standards was applied to the cartridge and the
Fig. 2. Chromatograms obtained with the blank (A) and the standard at 0.125 Ag/ml warfarin concentration (B). AU, absorbance units. The number above the peak identifies warfarin and corresponds to the retention time.
R. Lombardi et al. / Thrombosis Research 111 (2003) 281–284
283
the samples containing warfarin at 0.125 Ag/ml and the blank sample are shown in Fig. 2. The between-run reproducibility of the assay, which was estimated as coefficient of variation (CV) by repeat measurements (n = 11) on a plasma sample (warfarin concentration = 1.29 Ag/ml) from a patient on warfarin therapy, was 11.8%. Recovery, estimated by measuring warfarin concentration in five different assays for a drug-free plasma sample pooled from healthy subjects and spiked with warfarin at the final concentration of 2.5 Ag/ml, was 99 F 10.2. The coefficients of correlation between warfarin concentration and the INR or the weekly dosage for 50 treated patients were 0.55 ( p < 0.0001) or 0.25 ( p = 0.079) (Fig. 3).
4. Discussion
Fig. 3. Correlation between plasmatic warfarin concentration and international normalized ratio (INR) (r = 0.25, p = 0.079), or weekly warfarin dosage for 50 patients on oral anticoagulation (r = 0.55, p < 0.001).
unbound material was washed out with 1 ml 5% methanol in water followed by a second washing with 2% acetic acid in 5% methanol in water. The bound material was eluted by 1 ml of 2% ammonium hydroxide in 60% methanol in water. Eluted material was evaporated to dryness under a stream of nitrogen at 40 jC. The residue was eventually dissolved in 100 Al mobile phase and injected (20 Al) into the HPLC system (Waters). Separation was performed on a 3.9 150 mm symmetry shield column (RP8, 5 Am, Waters) and peaks were detected with a tunable absorbance UV detector (Waters) at 280 nm. The eluent (25 mM potassium phosphate, pH 7 with 45% methanol) was applied at a flow rate of 1 ml/min (isocratic conditions). On each test occasion, we run (in duplicate) a set of standards (ranging from 5 to 0.125 Ag/ml) for every seven unknown samples. Peak heights for standards and test plasma were measured and the concentration of warfarin was interpolated from the calibration curve (peak height vs. warfarin concentration).
Methods so far described are based on extraction of warfarin from plasma followed by gas chromatography or reversed-phase HPLC [3 –6]. Extraction is the crucial step and may be performed either in liquid- or solid-phase, which requires careful conditions to ensure good recovery of warfarin before sample injection. The solid-phase extraction requires the preparation of suitable columns, which in addition to the necessary expertise may make the entire assay procedure time-consuming and the sample preparation highly variable. We investigated the suitability of the readyfor-use, commercially available, miniature cartridges for sample preparation in the measurement of warfarin by HPLC. The method is reliable and easy to perform for those laboratories that have access to HPLC equipment. We investigated with this method patients on oral anticoagulants to assess correlation between the concentration of warfarin and the INR or the weekly dosage administered to the patients. Interestingly, the warfarin concentration was more correlated with the weekly dosage than with the INR. This is not surprising. Interaction with other drugs, dependency from diet, seasonal variation and age make the INR poorly dependent on warfarin concentration. This strengthens the concept that warfarin measurement alone would be of little help for dose-adjustment in patients on oral anticoagulants. However, this measurement may be useful in managing patients and should be included in the laboratory armamentarium of oral anticoagulant clinics. In conclusion, the method described proved to be easy to do, reproducible and specific for warfarin. It does not require sophisticated equipment other than the regular HPLC apparatus that is available in clinical laboratories.
3. Results
References
A typical calibration curve (peak height as absorbance unit vs. warfarin concentration) is shown in Fig. 1. Linearity was excellent (r2 = 0.999). Chromatograms correspondent to
[1] Hirsh J, Dalen JE, Anderson DR, Poller L, Bussey H, Ansell J, et al. Oral anticoagulants: mechanism of action, clinical effectiveness, and optimal therapeutic range. Chest 2001;119:8S – 21S. [2] WHO Expert Committee on Biological Standardization. Guidelines for
284
R. Lombardi et al. / Thrombosis Research 111 (2003) 281–284
thromboplastins and plasma used to control oral anticoagulant therapy. Technical Report Series 1999;889:48th report. Geneva, Switzerland. [3] Fasco MJ, Piper LJ, Kaminsky LS. Biochemical applications of a quantitative high-pressure liquid chromatographic assay of warfarin and its metabolites. J Chromatogr 1997;131:365 – 73. [4] Banfield C, Rowland M. Stereospecific fluorescence high-performance liquid chromatographic analysis of warfarin and its metabolites in plasma and urine. J Pharm Sci 1984;73:1392 – 6.
[5] Bush ED, Low LK, Trager WF. A sensitive and specific stable isotope assay for warfarin and its metabolites. Biomed Mass Spectrom 1983;10: 395 – 8. [6] Lang D, Bocker R. Highly sensitive and specific high-performance liquid chromatographic analysis of 7-hydroxywarfarin, a marker for human cytochrome P-4502C9 activity. J Chromatogr B, Biomed Appl 1995;672:305 – 9.