The aim of this study is to develop a specific, sensitive, rapid, and simple HPLC method with UV detection to study pharmacokinetics of tirofiban in serum of rats alone or in the presence of heparin as simultaneous therap

Arsova-Sarafinovska, Zorica

Balkanov, Trajan

Darkovska-Serafimovska, Marija

Gjorgoski, Icko

Janevik-Ivanovska, Emilija

Ugresic, Nenad

UGD Academic Repository

                                          DETERMINATION OF TIROFIBAN IN SERUM USING                       LIQUID CHROMATOGRAPHY WITH UV DETECTION    INTRODUCTION  The aim of this study is to develop a specific, sensitive, rapid, and simple HPLC method with UV detection to study pharmacokinetics of tirofiban in serum of rats alone or in the presence of heparin as simultaneous therapy. Thirty six Wistar rats were used randomly assigned in two groups: control group (n = 18), and group with venous thrombosis experimentally induced by ligature of the femoral vein (n = 18). Tirofiban hydrochloride was dissolved in sterile saline. The i.v. bolus doses (0.6 mg kg-1; 0.8 mg kg-1; 1 mg kg-1) were injected using tail vein. Blood samples were taken in the presence of heparin during one hour: starting 15 minutes after injection in the intervals of 15 minutes.  EXPERIMENTAL  Blood samples were centrifuged 5 min at 3500 rpm min-1. The serum was treated carefully, then methanol was added for precipitation of proteins (in ratio serum: methanol = 1:3). The tubes were again centrifuged 5 min at 3500 rpm min-1 and supernatant was injected into the HPLC column. HPLC separation was carried out at ambient temperature, using reversed-phase LiChrospher® 100 RP-18 column (4.0 mm × 250 mm, 5 μm particle size). The chromatographic separation was performed by isocratic mode with of 0,1 M KH2PO4 (pH = 5.0, adjusted with 1.0 N sodium hydroxide) and acetonitrile in the ratio of 80:20 % v/v as mobile phase with a flow rate of 1.0 ml min-1. UV detection was performed at 274 nm. The injection volume was 50 µl. Tirofiban concentration was also determined in spiked human and rat serum samples. System suitability was checked by evaluating different parameters (retention time, tailing factor, capacity factor, resolution, and selectivity).    CONCLUSION  The proposed RP-HPLC method is simple, accurate, precise, and rapid for the determination of tirofiban in serum and for monitoring its concentration in serum. The described method can be readily applied, without any interference from endogenous substances, to study pharmacokinetics of tirofiban in serum given alone or in the presence of heparin as simultaneous therapy and for therapeutic monitoring of levels of tirofiban in serum samples. RESULTS AND DISCUSSION   Tailing and capacity factors were obtained as 1.17 and 2.41 for tirofiban. Resolution factor for the system for tirofiban and heparin was 3.90. The retention times of tirofiban in methanol, human and rat serum samples were 9.1, 9.2, and 9.16 min, respectively (Figure 1.). The variation in retention time of tirofiban among five replicate injections of standard solution in methanol, human and rat serum samples was very little, giving relative standard deviations (RSD%) of  0.61%, 0.93%, and 0.82%, respectively.  The response was linear over the range of 0.03 – 0.18 mg mL-1 in mobile phase and serum samples. The limit of detection (LOD) for tirofiban was 1.84, 13,8 and 14.6 μg mL-1 in methanol, spiked rat serum and spiked human serum, respectively (Table 1.). Plasma concentration-time profile obtained from the experimental group was significantly different from plasma concentration-time profile obtained from the control group.                 M. Darkovska-Serafimovska 1,5,  Z. Arsova-Sarafinovska 2 ,  T. Balkanov3,  I. Gjorgoski 4,  N. Ugresic 5,   E. Janevik-Ivanovska 1     1,5. University Goce Delcev, Faculty of Medical Sciences, Krste Misirkov bb, 2000 Stip, Macedonia        2. Institute for Public Health of the Republic of Macedonia, 50 Divizija 6, 1000 Skopje, Macedonia       3. University Ss. Cyril and Methodius, Faculty of Medicine, 50 Divizija 6, 1000 Skopje, Macedonia       4. University Ss. Cyril and Methodius, Faculty of Natural Sciences and Mathematics, Gazi Baba bb,   1000  Skopje, Macedonia       5. University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia                       Characteristics of the linear regression analysis of tirofiban Chromatogram of standard solution of  tirofiban and heparin  in methanol (a) and chromatogram of serum spiked with   tirofiban and heparin (b) 

Determination of tirofiban in serum using liquid chromatography with UV detection

The aim of this study is to develop a specific, sensitive, rapid, and simple HPLC method with UV detection to study pharmacokinetics of tirofiban in serum of rats alone or in the presence of heparin as simultaneous therapy. Thirty six Wistar rats were used randomly assigned in two groups: control group (n = 18), and group with venous thrombosis experimentally induced by ligature of the femoral vein (n = 18). Tirofiban hydrochloride was dissolved in sterile saline. The i.v. bolus doses (0.6 mg kg-1; 0.8 mg kg-1; 1 mg kg-1) were injected using tail vein. Blood samples were taken in the presence of heparin during one hour: starting 15 minutes after injection in the intervals of 15 minutes. Tirofiban concentration was analysed immediately. Blood samples were centrifuged 5 min at 3500 rpm min-1. The serum was treated carefully, then methanol was added for precipitation of proteins (in ratio serum: methanol = 1:3). The tubes were again centrifuged 5 min at 3500 rpm min-1 and supernatant was injected into the HPLC column. HPLC separation was carried out at ambient temperature, using reversed-phase LiChrospher® 100 RP-18 column (4.0 mm × 250 mm, 5 μm particle size). The chromatographic separation was performed by isocratic mode with of 0,1 M KH2PO4 (pH = 5.0, adjusted with 1.0 N sodium hydroxide) and acetonitrile in the ratio of 80:20 % v/v as mobile phase with a flow rate of 1.0 ml min-1. UV detection was performed at 274 nm. The injection volume was 50 µl. Tirofiban concentration was also determined in spiked human and rat serum samples. System suitability was checked by evaluating different parameters (retention time, tailing factor, capacity factor, resolution, and selectivity). Tailing and capacity factors were obtained as 1.17 and 2.41 for tirofiban. Resolution factor for the system for tirofiban and heparin was 3.90. The retention times of tirofiban in methanol, human and rat serum samples were 9.1, 9.2, and 9.16 min, respectively (Figure 1.). The variation in retention time of tirofiban among five replicate injections of standard solution in methanol, human and rat serum samples was very little, giving relative standard deviations (RSD%) of  0.61%, 0.93%, and 0.82%, respectively.  The response was linear over the range of 0.03 – 0.18 mg mL-1 in mobile phase and serum samples. The limit of detection (LOD) for tirofiban was 1.84, 13,8 and 14.6 μg mL-1 in methanol, spiked rat serum and spiked human serum, respectively (Table 1.). Plasma concentration-time profile obtained from the experimental group was significantly different from plasma concentration-time profile obtained from the control group. The proposed RP-HPLC method is simple, accurate, precise, and rapid for the determination of tirofiban in serum and for monitoring its concentration in serum. The described method can be readily applied, without any interference from endogenous substances, to study pharmacokinetics of tirofiban in serum given alone or in the presence of heparin as simultaneous therapy and for therapeutic monitoring of levels of tirofiban in serum samples

DETERMINATION OF TIROFIBAN IN SERUM USING LIQUID CHROMATOGRAPHY WITH UV DETECTION  M. Darkovska-Serafimovska 1,5,  Z. Arsova-Sarafinovska 2 ,  T. Balkanov3,  I. Gjorgoski 4,  N. Ugresic 5,             E. Janevik-Ivanovska 1  1. University Goce Delcev, Faculty of Medical Sciences, Krste Misirkov bb, 2000 Stip, Macedonia  2. Institute for Public Health of the Republic of Macedonia, 50 Divizija 6, 1000 Skopje, Macedonia 3. University Ss. Cyril and Methodius, Faculty of Medicine, 50 Divizija 6, 1000 Skopje, Macedonia 4. University Ss. Cyril and Methodius, Faculty of Natural Sciences and Mathematics, Gazi Baba bb, 1000          Skopje, Macedonia 5. University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia  The aim of this study is to develop a specific, sensitive, rapid, and simple HPLC method with UV detection to study pharmacokinetics of tirofiban in serum of rats alone or in the presence of heparin as simultaneous therapy. Thirty six Wistar rats were used randomly assigned in two groups: control group (n = 18), and group with venous thrombosis experimentally induced by ligature of the femoral vein (n = 18). Tirofiban hydrochloride was dissolved in sterile saline. The i.v. bolus doses (0.6 mg kg-1; 0.8 mg kg-1; 1 mg kg-1) were injected using tail vein. Blood samples were taken in the presence of heparin during one hour: starting 15 minutes after injection in the intervals of 15 minutes. Tirofiban concentration was analysed immediately. Blood samples were centrifuged 5 min at 3500 rpm min-1. The serum was treated carefully, then methanol was added for precipitation of proteins (in ratio serum: methanol = 1:3). The tubes were again centrifuged 5 min at 3500 rpm min-1 and supernatant was injected into the HPLC column. HPLC separation was carried out at ambient temperature, using reversed-phase LiChrospher® 100 RP-18 column (4.0 mm × 250 mm, 5 μm particle size). The chromatographic separation was performed by isocratic mode with of 0,1 M KH2PO4 (pH = 5.0, adjusted with 1.0 N sodium hydroxide) and acetonitrile in the ratio of 80:20 % v/v as mobile phase with a flow rate of 1.0 ml min-1. UV detection was performed at 274 nm. The injection volume was 50 µl. Tirofiban concentration was also determined in spiked human and rat serum samples. System suitability was checked by evaluating different parameters (retention time, tailing factor, capacity factor, resolution, and selectivity). Tailing and capacity factors were obtained as 1.17 and 2.41 for tirofiban. Resolution factor for the system for tirofiban and heparin was 3.90. The retention times of tirofiban in methanol, human and rat serum samples were 9.1, 9.2, and 9.16 min, respectively (Figure 1.). The variation in retention time of tirofiban among five replicate injections of standard solution in methanol, human and rat serum samples was very little, giving relative standard deviations (RSD%) of  0.61%, 0.93%, and 0.82%, respectively.  The response was linear over the range of 0.03 – 0.18 mg mL-1 in mobile phase and serum samples. The limit of detection (LOD) for tirofiban was 1.84, 13,8 and 14.6 μg mL-1 in methanol, spiked rat serum and spiked human serum, respectively (Table 1.). Plasma concentration-time profile obtained from the experimental group was significantly different from plasma concentration-time profile obtained from the control group. The proposed RP-HPLC method is simple, accurate, precise, and rapid for the determination of tirofiban in serum and for monitoring its concentration in serum. The described method can be readily applied, without any interference from endogenous substances, to study pharmacokinetics of tirofiban in serum given alone or in the presence of heparin as simultaneous therapy and for therapeutic monitoring of levels of tirofiban in serum samples.   Table 1.  Characteristics of the linear regression analysis of tirofiban            Figure 1. Chromatogram of standard solution of  tirofiban and heparin in methanol (a) and chromatogram of serum spiked with tirofiban and heparin (b)  References  Oertel R, Köhler A, Koster A, Kirch W., Determination of Tirofiban in human serum by liquid chromatography-tandem mass spectrometry, J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Jun 5;805(1):181-5. 

Determination of tirofiban in serum using liquid chromatography with UV detection

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