On-line coupling of anion exchange and ion-pair chromatography for measurement of intracellular triphosphate metabolites of reverse transcriptase inhibitors.
We developed an automated on-line weak anion exchange (WAX) solid-phase extraction (SPE) method coupled with ion-pair (IP) chromatography-tandem mass spectrometry (MS/MS) detection for quantitatively measuring triphosphorylated metabolites of three reverse transcriptase inhibitors (RTI). The administered pro-drugs were Tenofovir disoproxil fumarate (TDF), Emtricitabine (FTC) and Lamivudine (3TC). Their intracellular metabolites Tenofovir-diphosphate (TFV-DP), Emtricitabine-triphosphate (FTC-TP), and Lamivudine-triphosphate (3TC-TP) were measured in peripheral blood mononuclear cells (PBMC). We coupled the WAX and IP chromatography systems using a combination of 6-port and 10-port switching valves, and we mixed the WAX elute with 1,5-dimethyl-hexyl-amine before IP chromatography separation. Multiple waste outlets allowed for eliminating potential matrix components interfering with MS/MS detection. Limits of detection were 9, 200 and 75 pg per sample for TFV-DP (448/176 m/z), FTC-TP (488/130 m/z) and 3TC-TP (468/119 m/z), respectively.
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341,United States.
Zsuzsanna
Z
Kuklenyik
Amy
A
Martin
Chou-Pong
CP
Pau
Angela
A
Holder
Ae S
AS
Youngpairoj
Qi
Q
Zheng
Mian-Er
ME
Cong
J Gerardo
JG
Garcia-Lerma
Walid
W
Heneine
James L
JL
Pirkle
John R
JR
Barr
D000225:Adenine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000068679:Emtricitabine; D006801:Humans; D019259:Lamivudine / Q000737:chemistry / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D063065:Organophosphonates / Q000737:chemistry / Q000378:metabolism; D011122:Polyphosphates / Q000737:chemistry* / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000068698:Tenofovir
D063065:Organophosphonates; D011122:Polyphosphates; D018894:Reverse Transcriptase Inhibitors; D003841:Deoxycytidine; D019259:Lamivudine; D000068698:Tenofovir; D000068679:Emtricitabine; D000225:Adenine