Abstract

Simultaneous analysis of doping drugs in human plasma and urine using HPLC- DAD

Author(s): LailaAbdel Fattah, AmiraM.El-Kosasy, OmarAbd El-Aziz, Naglaa Ebrahim

AhighlysensitiveRP-HPLCmethod has been developedfor simultaneous separation and quantitation of seven doping drugs, includingfour diuretics ‘Hydrochlorothiazide (HCTZ), Furosemide (FUR), Indapamide (IDP) and spironolactone (SPIRO)Â’, Salbutamol (SAL) as â-agonist, Testosterone (TSE) as anabolic and Betamethasone (BMS) as corticosteroid in spiked human plasma and urine, by usingZorbaxeclipseHC-C18column(250mmx4.6mmx5µm) with mobile phase acetonitrile: phosphoric acid pH3 (50:50, v/v)under isocratic conditionswith flow rate of 1.0mlmin-1 and at roomtemperature.Diode array detectorwas adjusted at ‘225, 272, 235, 242 and 244Â’ and 239 nm for quantitative determination of ‘HCTZ, SAL, FUR, IDPandTSEÂ’ and both ‘SPIRO and BMSÂ’, respectively. The linearity range for the studied drugs in the plasmawas 100-9000, 100-1800, 100-5000, 200-9000 and 1000-9000 ng.ml-1for ‘HCTZ and SALÂ’, for ‘FUR and TSEÂ’, for IDP, for SPIROand forBMS, respectively. LODs and LOQs valueswere found to be ‘31.16, 29.99, 28.14, 29.84, 31.98, 28.55 and 250.99Â’ and ‘94.42, 90.88, 85.27, 90.42, 96.91, 86.52 and 760.58Â’ ng ml-1 forHCTZ, SAL, FUR, IDP,TSE, SPIROandBMS, respectively.Also; theinvestigated drugs could bedetermined in spiked urinesamples after directdilutionandsolidphase extraction(SPE),where in the last way (SPE) HCTZ and SAL could not be determined, since they give irreproducible results. Indirectdilutionway; the linearityrangewas 150– 5000, 50 – 5000, 150 – 1500 and 100 – 5000 ng.ml-1 for ‘HCTZ and BMSÂ’, ‘SAL, IDP and TSEÂ’, ‘FURÂ’ and for ‘SPIROÂ’, respectively and the ‘LODs and LOQs-values Â’ were ‘39.41, 11.98, 35.52, 12.70, 14.11, 29.01 and 40.72Â’ and ‘119.42, 36.30, 107.64, 38.48, 42.76, 87.91 and 123.39Â’ ng.ml-1 forHCTZ, SAL, FUR, IDP,TSE, SPIROandBMS, respectively. In SPEmethod; the linearity rangewas 250-3000, 150- 6000 and 150-7000 ng.ml-1 for FUR, IDP and for ‘TSE, SPIRO and BMSÂ’, respectively and the ‘LODs and LOQs-valuesÂ’ were ‘70.11, 39.15, 42.71, 45.91 and 49.01Â’ and 212.45, 118.64, 129.42, 139.12 and 148.52 ng.ml- 1 for FUR, IDP, TSE, SPIROand BMS, respectively. It was shown that SPE is more sensitive, for determination ofFUR, IDP,TSE, SPIROandBMS, than direct dilution(only1:4dilutioncomparedto1:50folddilutionindirectdilution),however, HCTZandSALcould not be determined.