Reversed-phase HPLC method calibration

Amantea and Narang [58] used a reversed-phase HPLC method for the quantitation of omeprazole and its metabolites. Plasma was mixed with the internal standard (the 5-methyl analog of omeprazole), dichlor-omethane, hexane, and 0.1 M carbonate buffer (pH 9.8). After centrifugation, the organic phase was evaporated to dryness and the residue was dissolved in the mobile phase [methanol-acetonitrile-0.025 M phosphate buffer of pH 7.4 (10 2 13)] and subjected to HPLC at 25 °C on a column (15 cm x 4.6 mm) of Beckman Ultrasphere C8 (5 ym) with a guard column (7 cm x 2.2 mm) of Pell C8 (30-40 /im). The mobile phase flow-rate was 1.1 ml/min with detection at 302 nm. The calibration graphs are linear for <200 ng/ml, and the limits of detection were 5, 10, and 7.5 ng/ml for omeprazole, its sulfone, and its sulfide, respectively. The corresponding recoveries were 96.42% and 96% and the coefficients of variation (n = 5 or 6) were 3.0-13.9%. 

Sultana et al. [88] developed a reversed-phase HPLC method for the simultaneous determination of omeprazole in Risek capsules. Omeprazole and the internal standard, diazepam, were separated by Shim-pack CLC-ODS (0.4 x 25 cm, 5 m) column. The mobile phase was methanol-water (80 20), pumped isocratically at ambient temperature. Analysis was run at a flow-rate of 1 ml/min at a detection wavelength of 302 nm. The method was specific and sensitive with a detection limit of 3.5 ng/ml at a signal-to-noise ratio of 4 1. The limit of quantification was set at 6.25 ng/ml. The calibration curve was linear over a concentration range of 6.25—1280 ng/ml. Precision and accuracy, demonstrated by within-day, between-day assay, and interoperator assays were lower than 10%. 

A reverse-phase HPLC method with ESI-MS detection to characterize the pharmacokinetic behavior of procarbazine, a cytotoxic chemotherapeutic agent used in the treatment of lymphomas and hrain tumors. The data are used in a phase I trial concentrations are measured in human plasma. The calibration curve is linear in the 0.5-50 ng/ml concentration range. Average recovery rate 102.9%. Lower limit of quantitation 0.5 ng/ml accmacy 105.2% interday precision 3.6% RSD sample volume 150 p,l. Interday precisions at widely different concentrations 97-98%. The stability of the drug under storage and sample preparation conditions have also been thoroughly tested. Sensitivity is sufficient for monitoring plasma levels after oral administration. 

HPLC techniques have also been used in the determination of log P values. Lambert et al. (1990), for example, have described the development of a preformulation lipophilicity screen utilizing a C18 derivatized HPLC column. They appeared to prefer this column to the traditional reverse-phase HPLC columns, which may yield a poor correlation between log P and the capacity factor (k ). A potential problem with the use of HPLC retention data is that it is not a direct method and thus requires calibration. Futhermore, there may be problems with performing experiments above pH 8. 

Ma and Kim (1995) developed an on-line LC/TS/MS method for the analysis of PL molecular species in rat brain. After total lipid extraction, the extract was subjected to analysis with on-line reversed-phase HPLC and filament-on TS/MS. By using non-conventional HPLC conditions, partial separation of individual PL classes (PS, PI, PE and PC) and partial separation of molecular species within each class were achieved. By monitoring the retention time and the characteristic fragment ions (DG ions) formed in the filament-on TS process, individual molecular species in each PL class could be identified. Although non-linear calibration curves were observed for all DG ions monitored, even in the presence of an internal standard, semiquant-itative and quantitative results could still be obtained for a mixture of PLs. 

See Figure 8. HPLC equipment using reversed phase separation with fluorometric detection was used. Injection for correlation HPLC was accomplished with a newly developed device ( 4 ). The total range of phenol measured was five decades of concentration 0.01 -100 jug/L. The two higher concentrations (10 - 100 /i-g/L) were determined solely by conventional methods. The two lower concentrations (0.01 - 0.1 /xg/L) were determined by correlation HPLC with 16 and 3 sequences correlation time, respectively. Measurements at the 1 /xg/L level were carried out both by conventional and by correlation HPLC (1 sequence). The bars indicated on the calibration graph represent the peak area +3 (arbitrary units), where is the standard deviation... 

Zarghi et al. [76] developed an HPLC method, using a monolithic column, for quantification of omeprazole in plasma. The method is specific and sensitive with a quantification limit of 10 ng/ml. Sample preparation involves simple, one-step extraction procedure, and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 x 4.6 mm) column with an isocratic mobile phase consisting of 0.01 mol/1 disodium hydrogen phosphate buffer-acetonitrile (73 27) adjusted to pH 7.1. The wavelength was set at 302 nm. The calibration curve was linear over the concentration range 20-1500 ng/ml. The coefficients of variation for intra- and interday assay were found to be less than 7%. 

Franco et al. [45] described an HPLC method for simultaneous determination of the R-( ) and (S)-(+)-enantiomers of vigabatrin in human serum after precolumn derivatization with 2,4,6-trinitrobenzene sulfonic acid (TNBSA) and detection at 340 nm. Separation was achieved on a reversed phase chiral column (Chiralcel-ODR, 25 cm x 4.6 mm) using 0.05 M potassium hexafluorophosphate (pH4.5) acetonitrile ethanol (50 40 10) as a mobile phase at a flow rate of 0.9 ml/min. The calibration graphs for each enantiomer were linear over the concentration range of 0.5-40 fig/ml with a limit of quantification of 0.5 fig/ml. No interferences were found from commonly coadministered antiepileptic drugs. 

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