Simultaneous determination of 16 anti-HIV drugs in human plasma by high-performance liquid chromatography.
Therapeutic drug monitoring (TDM) is pivotal to improve the management of HIV infection. Here, a HPLC-UV method has been developed to quantify simultaneously seven HIV protease inhibitors (amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir; PIs), seven nucleoside reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine; NRTIs), and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine; NNRTIs) in human plasma. The volume of the plasma sample was 600 microL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 100 microL methanol. Twenty microliters of these samples were injected into a HPLC-UV system, the analytes were eluted on an analytical C(18) Symmetry column (250 mm x 4.6mm I.D.) with a particle size of 5 microm. The mobile phase (0.01 M KH(2)PO(4) and acetonitrile) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 35 min, the anti-HIV drugs were detected by UV at 240 and 260 nm. The calibration curves were linear up to 10 microg/mL. The absolute recovery ranged between 88 and 120%. The in vitro stability of anti-HIV drugs (0.005-10 microg/mL) in plasma has been studied at 24.0 degrees C. On these bases, a two to four analyte method has been tailored to the individual needs of the HIV-infected patient. The HPLC-UV method here reported has been validated and is currently applied to monitor PIs, NRTIs, and NNRTIs in plasma of HIV-infected patients. It allows to monitor the largest number of anti-HIV drugs simultaneously, appearing useful in a routine laboratory, and represents an essential step to elucidate the utility of a formal therapeutic drug monitoring for the optimal follow-up of HIV-infected patients.
Notari
Stefania
S
Istituto Nazionale per le Malattie Infettive-I.R.C.C.S. Lazzaro Spallanzani, Via Portuense 292, I-00149 Rome, Italy.
Bocedi
Alessio
A
Ippolito
Giuseppe
G
Narciso
Pasquale
P
Pucillo
Leopoldo Paolo
LP
Tossini
Gianna
G
Donnorso
Raffaele Perrone
RP
Gasparrini
Francesco
F
Ascenzi
Paolo
P
D019380:Anti-HIV Agents / Q000097:blood* / Q000627:therapeutic use; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods*; D004359:Drug Therapy, Combination; D015658:HIV Infections / Q000188:drug therapy; D017320:HIV Protease Inhibitors / Q000097:blood; D006801:Humans; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000097:blood; D012680:Sensitivity and Specificity
D019380:Anti-HIV Agents; D017320:HIV Protease Inhibitors; D018894:Reverse Transcriptase Inhibitors