Agilent Analysis of Testosterones in Serum: Zorbax SPE C18 Cartridge
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1. g e d Analysis of Testosterones in Serum Zorbax SPE C18 Cartridge e e c ee o Pad Application Brief i e a Pharmaceutical Qunjie Wang Testosterone and related compounds are present in the bloodstream at various levels both naturally and as a result of medical treatment and occasionally due to misuse It is of interest to determine levels of testosterone like compounds in the bloodstream with minimal background from the matrix e g blood proteins As shown below it is possible to significantly reduce levels of matrix compounds through the use of a Zorbax SPE C18 cartridge The analytes of interest are then easily quantitated by reversed phase HPLC Recovery of testosterone acetate and propionate were very high see Recovery DADI A Sig 254 16 Ref 360 100 A TESTO BL D meal ie o PP a Proteins A CH 607 Testosterone Testosterone m ARAR propionate Testosterone l f enanthate 204 W Merr J ie SEDI A Sig 254 16 Ref 360 100 A TESTOALDT s 2 izil mAU x 1205 il os Proteins B 1004 80 4 ol Testosterone acetate Testosterone ol propionate Testosterone E A enal te 204 rn od J Now FAN i ne T T T T T 0 2 4 6 8 Figure 1 Comparison of spiked serum sample before panel A and after panel B extraction using the Zorbax SPE C18 cartridge Notice effectiveness of the extraction In panel A the large amount of pro2. of goodness of technique as well as quality of the SPE packing For best results solutions should not be drawn completely through the cartridge until the final elute step Dr Qunjie Wang is a research chemist at Agilent Technologies based in Wilmington Delaw are For more information on our products and services please contact your local Agilent Technologies office or your local authorized Agilent distributor or visit our website at www agilent com chem Copyright 2001 Agilent Technologies All Rights Reserved Reproduction adaptation or translation without prior w ritten permission is prohibited except as allow ed under the copyright laws Publication Number 5988 2530EN ope Agilent Technologies
3. tein in the sample interferes with optimal quantitation of testosterone propionate Once the sample has passed through the cartridge analytes are clearly detected and quantifiable panel B Testosterone enanthate is included in samples as an internal standard Solid Phase Extraction Method CARTRIDGE 100 mg 1 ml Zorbax SPE C18 P N 5184 3590 Box of 100 CONDITION 1 0 mL Methanol 1 0 mL Deionized Water LOAD 1 0 mL Sample see sample preparation WASH 1 0 mL 5 Methanol in Water ELUTE 1 0 mL Methylene Chloride M ethanol 50 50 viv EVAPORATE and RECONSTITUTE Room Temperature nitrogen stream Add 200 uL of Methanol with 10 pL 100 pg mL testosterone enanthate as internal standard Sample Preparation Testosterone acetate and testosterone propionate 1mg mL each in Methanol Spike 10 uL above solution with 8 mL bovine serum 2 mL Deionized Water Internal Standard Prepare a 1 mg ml solution of testosterone enanthate in Methanol 42i Agilent Technologies HPLC Method LC Instrument Agilent 1100 Column Zorbax XDB C8 4 6 mmx 75 mm 3 5 um P N 966967 906 Eluent A Acetonitrile Water 60 40 B Acetonitrile Gradient 0 B 60 B in 12 min UV 254 nm Flow 1 5 mL min Temperature 25 C Inj Vol 20 uL Recovery Testosterone Acetate 1 pg mL X 95 n 4 Testosterone Propionate 1 pg mL X 92 n 4 Recovery of analyte in these experiments is an indication
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Agilent Analysis of Testosterones in Serum: Zorbax SPE C18 Cartridge