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Sample Preparation	Solid Phase Extraction
Sample/Matrix	Urine spiked with drugs and metabolites at 300 ng/mL
SPE Well Plate	Supel-Select HLB 96-well plate, 60 mg/well (575662-U)
Condition	1 mL methanol followed by 1 mL water
Sample Addition	1 mLhydrolyzed urine
Washing	1 mL water followed by 1 mL 25% methanol
Elution	1 mL acetonitrile
Column	Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 µm particles (53569-U)
Column Temp.	35 °C
Mobile Phase	[A] 5 mM ammonium formate, pH 4.2 (adjusted with formic acid); [B] 5 mM ammonium formate in (95:5 acetonitrile:water) pH 5.2 (adjusted with formic acid)
Gradient	15% B held for 1 min, to 70% B in 2 min, held at 70% B for 7 min
Flow Rate	0.4 mL/min
Sample	300 ng/mL
Injection	3 µL
Detector	ESI(+) TOF, full scan, m/z 100-1000

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Analysis Note	The workflow for the LC/MS analysis of native drugs and associated metabolites from urine is shown here. The convenient liquid form of beta-glucuronidase from Sigma was used for hydrolysis of the glucuronide metabolites. SPE using Supel-Select HLB yielded clean extracts. The Ascentis Express F5 Columns gave highly efficient, well-resolved peaks and rapid separation. Ultra high purity LC-MS solvents and additives provided trouble-free operation, and Cerilliant CRMs gave reliable quantification., Sample pretreatment: β-glucuronidase hydrolysis
Categories	Analytical Chromatography, SPE Applications, Clinical, Forensics and Toxicology, catecholamines, Opiate, Opiates
Featured Industry	Clinical \r\nForensics and Toxicology
Legal Information	Ascentis is a registered trademark of Sigma-Aldrich Co. LLC, Supel is a trademark of Sigma-Aldrich Co. LLC
Suitability	Application for SPE, application for HPLC

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