FDA Bioanalytical Method Validation Research Articles

Development of liquid chromatographic method for fosinoprilat determination in human plasma using microemulsion as eluent

Fosinopril sodium presents a prodrug for the active angiotensin converting enzyme (ACE) inhibitor, fosinoprilat. The dual elimination of fosinoprilat by the liver and the kidney distinguishes fosinopril from other angiotensin converting enzyme inhibitors. Such ways of elimination are important for antihypertensive therapy of patients on haemodialysis. The paper presents development and evaluation of a new and sensitive liquid chromatographic (LC) method for the analysis of fosinoprilat in plasma obtained from patients on haemodialysis. A microemulsion system mixture as mobile phase has been used for the separation and analysis of fosinoprilat in plasma samples. The plasma samples were injected directly onto the HPLC system (Waters Breeze) after appropriate sample dilution with mobile phase. Separations were performed on the Bakerbond ENV 4.6 mm × 150 mm, 5 μm particle size column with UV detection at 220 nm. The flow rate was 1.00 mL min −1. The mobile phase consisted of 1.0% (w/v) of diisopropyl ether, 2.0% (w/v) of sodium dodecyl sulphate (SDS), 6.0% (w/v) of n-propanol and 91% (w/v) of aqueous 25 mM di-sodium hydrogen phosphate, pH adjusted to 2.8 with 85% orthophosphoric acid. The developed method was then subjected to method validation according to the criteria stated in the FDA bioanalytical method validation guidance. The results for specificity, linearity, low limit of quantification (LLOQ), precision, accuracy and stability were within the accepted criteria. The unique approach applied in this paper makes possible the determination of fosinoprilat even in the presence of metabolites of other drugs, so the method can be used for obtaining the reliable results in a fast and simple way.

Validation of the anthrax lethal toxin neutralization assay

A validation of the performance characteristics of a toxin neutralization assay is presented. This in vitro assay measures the functional ability of antisera, containing antibodies to anthrax lethal toxin, to specifically protect J774A.1 cells against Bacillus anthracis lethal toxin cytotoxicity. This colormetric assay is based upon the reduction of MTT by living cells. Human and rabbit antisera produced against anthrax vaccine absorbed (AVA) were used to validate the assay. Results showed a high level of repeatability and reproducibility, particularly for a bio-assay. Inter-assay variability in absorbance values was the most prominent negative finding however, an acceptable level was demonstrated with a ratio [neutralization ratio (NR)] of the test serum 50% effective dose (ED 50) to the reference standard ED 50. Accuracy was maintained, even in samples with minimal neutralizing capacity, and linearity was noted when sample dilutions resulted in accurate prediction of the Y maxand Y min. Specificity tests demonstrated that normal sera did not have an observable effect on the ability of the reference standard to neutralize toxin. The assay remained stable against time, temperature, and freeze/thaw effects on the reference standards, but not on the toxin. The assay also remained stable against media and solution storage effects. Cell passage number and cell plating density were two critical parameters identified during the robustness studies that may be responsible for inter-assay variability in absorbance values. The work was performed in accordance with the FDA's Bioanalytical Method Validation Guidance for Industry and the FDA's Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58).

Determination of glimepiride in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS–MS) method has been developed at our center for the determination of glimepiride in human plasma. After the addition of the internal standard, plasma samples were extracted by liquid–liquid extraction technique using diethyl ether. The compounds were separated on a prepacked C 18 column using a mixture of acetonitrile, methanol and ammonium acetate buffer as mobile phase. A Finnigan LCQ DUO ion trap mass spectrometer connected to an Alliance Waters HPLC was used to develop and validate the method. The analytical method was validated according to the FDA bioanalytical method validation guidance. The results were within the accepted criteria as stated in the aforementioned guidance. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 5.0–500.0 ng/ml with a coefficient of determination ( r 2) of 0.9998. Accuracy for glimepiride ranged from 100.58 to 104.48% at low, mid and high levels. The intra-day precision was better than 12.24%. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 5.0 ng/ml with a precision of 7.96%. The proposed method enables the unambiguous identification and quantitation of glimepiride for pharmacokinetic, bioavailability or bioequivalence studies.

Determination of loratadine in human plasma by liquid chromatography electrospray ionization ion-trap tandem mass spectrometry

A sensitive and specific liquid chromatography electrospray ionization ion-trap mass spectrometry (LC–ESI-IT-MS/MS) method has been developed and validated for the identification and quantitation of loratadine in human plasma. After the addition of the internal standard (IS), plasma samples were extracted using isooctane:isoamyl alcohol mixture. The compounds were separated on a prepacked Zorbax phenyl column using a mixture of acetonitrile, 0.20% formic acid as mobile phase. A Finnigan LCQ DUO ion-trap mass spectrometer connected to a Waters Alliance high performance liquid chromatography (HPLC) was used to develop and validate the method. The results were within the accepted criteria as stated in the FDA bioanalytical method validation guidance. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 0.10–10.0 ng/ml with a coefficient of determination ( r 2) of 0.9998. Accuracy for loratadine ranged from 105.00 to 109.50% at low, mid and high levels. The intra-day precision was better than 10.86%. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 0.10 ng/ml with a precision of 9.84%. The proposed method enables the unambiguous identification and quantitation of loratadine for pharmacokinetic, bioavailability or bioequivalence studies.