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Sample Preparation and Extraction in Small Sample Volumes Suitable for Pediatric Clinical Studies: Challenges, Advances, and Experiences of a Bioanalytical HPLC-MS/MS Method Validation Using Enalapril and Enalaprilat.

Burckhardt BB, Laeer S - Int J Anal Chem (2015)

Bottom Line: Enalapril, enalaprilat, and benazepril served as sample drugs.The applied sample preparation and extraction successfully reduced the absolute and relative matrix effect to comply with international guidelines.Recoveries ranged from 77 to 104% for enalapril and from 93 to 118% for enalaprilat.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-University, 40225 Düsseldorf, Germany.

ABSTRACT
In USA and Europe, medicines agencies force the development of child-appropriate medications and intend to increase the availability of information on the pediatric use. This asks for bioanalytical methods which are able to deal with small sample volumes as the trial-related blood lost is very restricted in children. Broadly used HPLC-MS/MS, being able to cope with small volumes, is susceptible to matrix effects. The latter restrains the precise drug quantification through, for example, causing signal suppression. Sophisticated sample preparation and purification utilizing solid-phase extraction was applied to reduce and control matrix effects. A scale-up from vacuum manifold to positive pressure manifold was conducted to meet the demands of high-throughput within a clinical setting. Faced challenges, advances, and experiences in solid-phase extraction are exemplarily presented on the basis of the bioanalytical method development and validation of low-volume samples (50 μL serum). Enalapril, enalaprilat, and benazepril served as sample drugs. The applied sample preparation and extraction successfully reduced the absolute and relative matrix effect to comply with international guidelines. Recoveries ranged from 77 to 104% for enalapril and from 93 to 118% for enalaprilat. The bioanalytical method comprising sample extraction by solid-phase extraction was fully validated according to FDA and EMA bioanalytical guidelines and was used in a Phase I study in 24 volunteers.

No MeSH data available.


Related in: MedlinePlus

The plots show the accuracy results of 22 serum calibration curves and the accuracy results of 7 urinary calibration curves (each covering 11 concentration levels per drug substance) of enalapril (black) and enalaprilat (grey) used for the evaluation of the obtained results of the conducted Phase I study. Additionally the accuracy thresholds (dashed lines) according to FDA and EMA bioanalytical guidelines for all concentrations levels (±15%) and the LLOQ (±20%) are indicated.
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Related In: Results  -  Collection


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fig8: The plots show the accuracy results of 22 serum calibration curves and the accuracy results of 7 urinary calibration curves (each covering 11 concentration levels per drug substance) of enalapril (black) and enalaprilat (grey) used for the evaluation of the obtained results of the conducted Phase I study. Additionally the accuracy thresholds (dashed lines) according to FDA and EMA bioanalytical guidelines for all concentrations levels (±15%) and the LLOQ (±20%) are indicated.

Mentions: For the Phase I study, in total approximately 1600 serum and 600 urinary samples were analyzed. No clotting of serum sample solution in any SPE cavity was noticed. A shift in the retention times of enalapril, enalaprilat, and benazepril in purified serum samples of 24 different sources was not detected during the analysis by HPLC-MS/MS and allowed for an automated intergation. Furthermore, hemolyzed samples did not affect the extraction run negatively and showed no significant interference during analysis. The determined pharmacokinetic results of the Phase I study will be used to apply a new marketing authorization and remain therefore confidential. However, during sample determination 22 calibration curves in serum and 7 in urine were required to quantify the drug concentration in the corresponding samples. Obtained results of the calibration curves on intra- and interrun accuracy proved the applicability of the established bioanalytical method comprising the good sample extraction and their suitable preparation. Figure 8 shows the accuracy results for the serum and urine calibration curves.


Sample Preparation and Extraction in Small Sample Volumes Suitable for Pediatric Clinical Studies: Challenges, Advances, and Experiences of a Bioanalytical HPLC-MS/MS Method Validation Using Enalapril and Enalaprilat.

Burckhardt BB, Laeer S - Int J Anal Chem (2015)

The plots show the accuracy results of 22 serum calibration curves and the accuracy results of 7 urinary calibration curves (each covering 11 concentration levels per drug substance) of enalapril (black) and enalaprilat (grey) used for the evaluation of the obtained results of the conducted Phase I study. Additionally the accuracy thresholds (dashed lines) according to FDA and EMA bioanalytical guidelines for all concentrations levels (±15%) and the LLOQ (±20%) are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4383429&req=5

fig8: The plots show the accuracy results of 22 serum calibration curves and the accuracy results of 7 urinary calibration curves (each covering 11 concentration levels per drug substance) of enalapril (black) and enalaprilat (grey) used for the evaluation of the obtained results of the conducted Phase I study. Additionally the accuracy thresholds (dashed lines) according to FDA and EMA bioanalytical guidelines for all concentrations levels (±15%) and the LLOQ (±20%) are indicated.
Mentions: For the Phase I study, in total approximately 1600 serum and 600 urinary samples were analyzed. No clotting of serum sample solution in any SPE cavity was noticed. A shift in the retention times of enalapril, enalaprilat, and benazepril in purified serum samples of 24 different sources was not detected during the analysis by HPLC-MS/MS and allowed for an automated intergation. Furthermore, hemolyzed samples did not affect the extraction run negatively and showed no significant interference during analysis. The determined pharmacokinetic results of the Phase I study will be used to apply a new marketing authorization and remain therefore confidential. However, during sample determination 22 calibration curves in serum and 7 in urine were required to quantify the drug concentration in the corresponding samples. Obtained results of the calibration curves on intra- and interrun accuracy proved the applicability of the established bioanalytical method comprising the good sample extraction and their suitable preparation. Figure 8 shows the accuracy results for the serum and urine calibration curves.

Bottom Line: Enalapril, enalaprilat, and benazepril served as sample drugs.The applied sample preparation and extraction successfully reduced the absolute and relative matrix effect to comply with international guidelines.Recoveries ranged from 77 to 104% for enalapril and from 93 to 118% for enalaprilat.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-University, 40225 Düsseldorf, Germany.

ABSTRACT
In USA and Europe, medicines agencies force the development of child-appropriate medications and intend to increase the availability of information on the pediatric use. This asks for bioanalytical methods which are able to deal with small sample volumes as the trial-related blood lost is very restricted in children. Broadly used HPLC-MS/MS, being able to cope with small volumes, is susceptible to matrix effects. The latter restrains the precise drug quantification through, for example, causing signal suppression. Sophisticated sample preparation and purification utilizing solid-phase extraction was applied to reduce and control matrix effects. A scale-up from vacuum manifold to positive pressure manifold was conducted to meet the demands of high-throughput within a clinical setting. Faced challenges, advances, and experiences in solid-phase extraction are exemplarily presented on the basis of the bioanalytical method development and validation of low-volume samples (50 μL serum). Enalapril, enalaprilat, and benazepril served as sample drugs. The applied sample preparation and extraction successfully reduced the absolute and relative matrix effect to comply with international guidelines. Recoveries ranged from 77 to 104% for enalapril and from 93 to 118% for enalaprilat. The bioanalytical method comprising sample extraction by solid-phase extraction was fully validated according to FDA and EMA bioanalytical guidelines and was used in a Phase I study in 24 volunteers.

No MeSH data available.


Related in: MedlinePlus