Use of small sample drug level monitoring for paediatric PI concentrations

Polly Clayden, HIV i-Base

A poster from J Lam and coworkers from the University of Southern California (USC), Pharmacology, Los Angeles, USA reported their methodology for total drug analysis from plasma with protein precipitation. This methodology simultaneously analyses multiple protease inhibitors in a single run, with a smaller injection volume, making it easier to use TDM, particularly in paediatrics, than previously published assays.

The investigators added a volume of 100 uL of 500 ng/mL of SQV as the internal standard to 100 mcg/mL of each plasma standard. The entire sample was protein precipitated by adding 500 uL of acetonitrile and centrifuged at 13,000 rpm for 5 minutes. The supernatant was evaporated to dryness, reconstituted with 150 ug/mL of mobile phase (58%: [v/v] acetonitrile + 42% 20 mM NH4Acetate, pH4.5), and then centrifuged for 5 minutes at 13,000 rpm. An aliquot of 135 ug/mL of the supernatant was transferred into HPLC injection vials.

A volume of 10 uL per sample was injected and the concentration was determined using a Sciex API 3+ mass spectrometer coupled with an Agilent 1100 HPLC and well plate autosampler. The analytes were separated using a Hypurity C18 column (50 x 4.6 mm, 5 um), where the flow-rate was 0.4 mL/min to elute the PIs.

The investigators found the CVs for both intra and inter-day precision were <10% for each compound. Accuracy was measured at >85%. Retention times were 1.44, 2.57, 4.12, 4.28, and 4.35 minutes for ritonavir, saquinavir, nelfinavir, lopinavir, and atazanavir, respectively. The total sample run time was 7.2 minutes.

The mass transitions were 705.3 to 335.3, 568.2 to 330.2, 721.2 to 296.2, 629.4 to 447.6 and 671.3 to 570.3 for atazanavir, nelfinavir, ritonavir, lopinavir, and saquinavir, respectively.

This methodology was validated over the concentration ranges of 0.025-2.0 ug/mL, for all of the protease inhibitors.

The investigators explained that this validated LC/MS assay method uses a small sample volume with a low CV. Use of a small sample volume is advantageous, especially when TDM used for paediatric patients. They noted: “The ability to minimise the amount of blood extracted, handle small patient samples, and rapidly analyse them will enable TDM and pharmacokinetic studies to be used in the paediatric setting.”


Lam J, M. Neely JM, Bi L, Louie S et al. LC/MS analysis of protease inhibitors. 7th International Workshop on Clinical Pharmacology of HIV Therapy, 20-22 April 2006, Lisbon. Abstract 17.

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