Determination of eflornithine enantiomers in plasma by precolumn derivatization with o-phthalaldehyde-N-acetyl-L-cysteine and liquid chromatography with UV detection.

A bioanalytical method for indirect determination of eflornithine enantiomers in 75 microL human plasma has been developed and validated. L- and D-eflornithine were derivatized with o-phthalaldehyde and N-acetyl-L-cysteine to generate diastereomers which were separated on two serially connected Chromolith Performance columns (RP-18e 100 x 4.6 mm i.d.) by a isocratic flow followed by a gradient flow for elution of endogenous compounds. The diastereomers were detected with UV (340 nm). The between-day precisions for L- and D-eflornithine in plasma were 8.4 and 2.3% at 3 microm, 4.0 and 5.1% at 400 microm, and 2.0 and 3.7% at 1000 microm. The lower limit of quantification was determined to be 1.5 microm, at which precision was 14.9 and 9.9% for L- and D-eflornithine, respectively.

Determination of lamivudine, zidovudine, and nevirapine in capillary blood sampled on filter paper by LC.

A bioanalytical method for determination of lamivudine (3TC), zidovudine (AZT), and nevirapine (NVP) in 100 microL capillary blood applied onto sampling paper has been developed and validated. The antiretroviral drugs (ARV) were analyzed by reversed phase gradient liquid chromatography with UV detection. Separation was performed on a Zorbax SB C(8) (250 x 4.6 mm) column with a two-step gradient: (i) methanol-0.05 mol/L acetic acid-sodium acetate buffer (pH 3.95, 15:85 v/v) and (ii) methanol-0.05 mol/L acetic acid-sodium acetate buffer (pH 3.95, 50:50 v/v) with a flow rate of 1.0 mL/min. UV detection was performed at 260 nm. Total assay precisions were 6.3, 4.7, and 4.9% for 3TC at 0.34, 0.69, and 3.9 microg/mL, and 5.1, 5.5, and 3.2% for AZT at 0.40, 0.80, and 4.5 microg/mL. For NVP, total assay precisions were 5.2, 8.3, and 3.5% at 2.6, 4.5, and 8.8 microg/mL. Lower limit of quantifications (LLOQ) were 0.11 and 0.13 microg/mL for 3TC and AZT where the precisions were 2.0% for both the analytes. For NVP, LLOQ was 1.3 microg/mL where precision was 2.6%. Concentrations were determined for 10 h for two subjects receiving standard twice daily antiretroviral therapy containing 3TC, AZT, and NVP. Maximum 3TC concentrations were 2.5 and 2.8 microg/mL for subject 1 and 2, respectively. For AZT, maximum concentrations were 1.8 and 1.1 microg/mL while being 15 and 9.6 microg/mL for NVP. Pre-dose trough concentration of NVP was 11 microg/mL for subject 1 and 9.6 microg/mL for subject 2.

Development and validation of an automated solid-phase extraction and liquid chromatographic method for determination of lumefantrine in capillary blood on sampling paper.

A bioanalytical method for the determination of lumefantrine in 100 microl blood applied onto sampling paper, by solid-phase extraction and liquid chromatography, has been developed and validated. Whatman 31 ET Chr sampling paper was pre-treated with 0.75 M tartaric acid before sampling capillary blood to enable a high recovery of lumefantrine. Lumefantrine was extracted from the sampling paper, then further purified using solid-phase extraction and finally quantified with HPLC. The between-day variation was below 10% over the range 0.4-25 microM. The lower limit of quantification was 0.25 microM in 100 microl capillary blood. No decrease in lumefantrine concentration in dried blood spot is seen after 4 months storage at 22 degrees C. The method was also evaluated in field samples from patients in Tanzania after treatment with lumefantrine/artemether. Lumefantrine could be estimated accurately enough to assess bioavailability and treatment compliance on day 7 (i.e. 4 days after the last dose) after a standard regimen with the lumefantrine/artemether combination.

Determination of melatonin in saliva using automated solid-phase extraction, high-performance liquid chromatography and fluorescence detection.

A sensitive bioanalytical method for the determination of melatonin in saliva by solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and fluorescence detection has been developed and validated. Saliva was collected with a Salivette sampling device (Sarstedt) and a mixed-mode SPE column was used for the extraction of melatonin and internal standard (N-acetyl-6-methoxytryptamine) from the saliva. Chromatographic separation was performed using a HyPurity C18 LC column (150 x 2.1 mm) with mobile phase acetonitrile-ammonium hydrogen carbonate buffer, 0.015 M, pH 6.8 (23:77, v/v). Excitation and emission wavelengths were set to 285 nm and 345 nm, respectively. The within-day precision for the method at 50 pmol/L was 7.9 % and the between-day precision was 10.5 %. The limit of quantification was 50 pmol/L.

Determination of melatonin in human plasma with solid-phase extraction, high-performance liquid chromatography and fluorescence detection.

A new bioanalytical method for the determination of melatonin in plasma with high-performance liquid chromatography (HPLC) and fluorescence detection preceded by solid-phase extraction has been developed and validated. Melatonin was extracted from 3 mL plasma using a Waters Oasis HLB solid-phase extraction cartridge and the elute was evaporated to dryness and dissolved in 200 microl mobile phase; acetonitrile-phosphate buffer, 0.01 M pH 7.2 (25:75, v/v). 125 microL was injected into the HPLC system and separation was carried out on a Waters SymmetryShield RP18 column 5 microm (250 x 4.6 mm). Excitation and emission wavelengths were set to 285 nm and 345 nm, respectively. The HPLC system was able to separate melatonin and internal standard (5-fluorotryptamine) from other endogenous indole compounds such as serotonin and tryptophan. Determination down to 0.10 nmol/L was possible, with an intra-assay precision of about 13%. Melatonin was stable in plasma for at least 30 days at about 23 degrees C.