METHOD DEVELOPMENT OF HYDROPHILIC INTERACTION CHROMATOGRAPHY FOR SCREENING AND SEPARATION OF SEVERAL DIURETIC DOPING AGENTS IN URINE

• Main Author: Iswandi
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34405
• Institution: Institut Teknologi Bandung

Nowadays, the use of doping by athletes is increasing both the type and amount of substance. The athletes usually use doping to improving athletes' physical and psychological abilities. Either by using substances with an unusual amount or by prohibited method. Chemical substances used as doping have different pharmacological effects because they have different physicochemical properties therefore an analytical method is required for screening and identification of doping use atlet. In general, this study aims to study the characteristics of effectiveness and the development doping compounds screening method in urine using hydrophilic columns for the separation of polar compounds. This research was also conducted to find optimal conditions and validate the analysis method of diuretics in urine by hydrophilic interaction liquid chromatography using the Zic HILIC column. Diuretic compounds have different partition coefficients (log P) and pKa basically to choosing an analysis method. Partition coefficient (log P) is one of the physical chemical properties that is important to liphopphicilitas a subtances. The log P value of the diuretics are ranging from -0.3 to 3.7, this indicates that these substances are polar. Screening of diuretics were carried out by liquid chromatography using the Zic- HILIC column and UV detector. Observations for the recorded optimization were the ability to selectively separate analytes through of retention time (tR), selectivity factor (?), resolution (Rs) and sensitivity of the method through the determination limit of detection (LOD). The optimum condition the mobile phase composition of acetonitrile: buffer ammonium acetate (pH 5, 5 mM) with a ratio (95: 5), with a flow rate of 0.5 ml / minute to separation of (furosemide-hydrochlorothiazide),(furosemideindapamide), (spironolactone-bumetanide) ; (spironolactone-etacrinic acid) and (bumetanide-indapamide). This condition produced resolution value of 2.99 ; 3,37 ; 2,53 ; 2,86 ; and 2,82. Value of LOD furosemide, hidrochlortiaziade, indapamide, spironolactone, bumetanide, and etacrynic acid respectively is 0.79 ; 0.58 ; 0.66 ; 0.79 ; 0.32 and 0.58 ppm . Value of LOQ furosemide, hidrochlortiaziade, indapamide, spironolactone, bumetanide, and etacrynic acid respectively is 2.40 ; 1.75 ; 2.00 ; 0.49 ; 0.97 and 2.14 ppm. The optimum separation condition of furosemide and spironolactone was the mobile phase composition of acetonitrile: ammonium acetate buffer (pH 5.5 mM) with a ratio (90 : 10), with a flow rate of 0.5 ml / minute. With this condition, the value of resolution = 3.14. The value of LOD furosemide and spironolactone is 0.16 and 0.71 ppm . The value LOQ furosemide and spironolactone is 0.49 and 2.14 ppm. The optimum condition separation of hidroclortiazide, triamterene, and bendroflumetiazide was acetonitrile: ammonium acetate buffer solution (pH 5.0 ; 5.0 mM) ( 90:10) and flow rate of 0.5 ml / minute. With this condition, the value of resolution 3.75 and 2.85. Separation mixture bumetanide, indapamide and spironolactone was compotition acetonitrile: ammonium acetate buffer solution (pH 5.0 ; 5.0 mM) ( 95:5) and flow rate of 0.5 mL / minute. With this condition, the value of resolution 1.77 and 4.00. Separtion mixture furosemide, azetozolamide anda indapamide was compotition acetonitrile: ammonium acetate buffer solution (pH 5.0 ; 5.0 mM) ( 90:10) and flow rate of 0.75 mL / minute. With this condition, the value of resolution 1.86 and 3.13. Value of LOD, hidrochlortiaziade, bendroflumetiazide, bumetanide, indapamide, spironolactone, furosemide and azetozolamide respectively is 0.58 ; 1.05 ; 0.62 ; 0.32 ; 0.66 ; 0,16 ; 0.79 and 0.66 ppm. Value of LOQ, hidrochlortiaziade, bendroflumetiazide, bumetanide, indapamide, spironolactone, furosemide and azetozolamide respectively is 1.75 ; 3.18 ; 1.88 ; 0.97 ; 2.00 ; 0,49 ; 2.40 and 2.00 ppm. The optimum conditions separation of spironolactone, indapamide, bumetanide and furosemide, were acetonitrile: ammonium in acetate buffer (pH 6.0; 10.0 mM) (95: 5) with flow rate of 0.5 ml / minute. The resolution values obtained with this condition were 1.75; 5.43 and 2.23. Mixture of fusrosemide, azetozolamide, indapamide and hidroclortiazide obtained composition mobile phase acetonitrile: ammonium acetate buffer (pH 5.0; 5.0 mM) with ratio 90: 10 and flow rate of 0.5 ml / minute. The resolution values obtained with this condition were 3.00; 1.82 and 2.77. For the mixture etacrynic acid, bendroflumethiazide, hydrochlorothiazide and triamterene was mobile phase acetonitrile: ammonium acetate buffer (pH 6.0; 10.0 mM) with ratio 95: 5 and flow rate 1.0 ml / minute. The resolution values obtained from this condition were 3.75; 2.29 and 3.91. Value of LOD spironolactone, indapamide, bumetanide, furosemide, azetozolamide, hidrochlortiaziade, etacrynic acid, bendroflumethiaziade and triamterene respectively is 0.16 ; 0.66 ; 0.32 ; 0.79 ; 0.47 ; 0.58 ; 0,71 ; 0.62 and 1.05 ppm. Value of LOD respectively is 0.49 ; 2.00 ; 0.97 ; 2.40 ; 1.44 ; 1.75 ; 2.14 ; 1.88 and 3.18 ppm. The optimum conditions separation 5 compounds (etacrynic acid, spironolactone, indapamide, furosemide and bumetanide) obtained was acetonitrile: ammonium acetate (pH 5.0; 5.0 mM) with a ratio (95: 5) and flow rate of 0.5 ml /minute. The resolution values obtained with this condition were 1.25; 2.67; 3.20; and 1.81. The value LOD respectively is 0.71 ; 0.16 ; 0.66 ; 0.79 and 0.32 ppm. The value LOQ respectively is 2.14 ; 0.49 ; 2.00 ; 2,40 ; and 0.97 ppm. The optimum conditions separation 6 compounds ( furosemide, indapamide, spironolactone bendroflumethiaziade, azetozolamide and hidrochlortiaziade) obtained was composition acetonitrile: ammonium acetate (pH 6.0; 10.0 mM) with a ratio (95: 5) and flow rate of 0.5 ml /minute. The resolution values obtained with this condition were 1.18 ; 2.70 ; 0.80; 1.13 and 0.85. The value LOD respectively is 0.79 ; 0.66 ; 0.16 ; 0.62 ; 0.47 and 0.58 ppm. The value LOQ respectively is 2.40 ; 2.0 ; 0.49 ; 1.88 ; 1,44 ; and 1.75 ppm. Application methode HILIC in sample urine added by diuretic doping compounds using selected conditions showed good selectivity with the order of spironolactone, indapamide, bumetamide, furosemide, bendroflumetiazid, triamteren and hydrochlorothiazide with resolution> 1.5. The analytical method developed has accepted for requirements the validation criteria and can also be applied for the determination of diuretic doping compounds.