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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

Determined split peak in serum with the transition 349.1 → 206.1 m/z during method development. The split peak was measured on several HPLC columns after SPE purification by Oasis MCX. In grey, the ion count of a low enalaprilat concentration in serum is shown that clearly identifies the split peak. As reference, the enalaprilat standard solved in mobile phase is presented by the black line without any split peak (base line is nudged to prevent overlap).
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fig3: Determined split peak in serum with the transition 349.1 → 206.1 m/z during method development. The split peak was measured on several HPLC columns after SPE purification by Oasis MCX. In grey, the ion count of a low enalaprilat concentration in serum is shown that clearly identifies the split peak. As reference, the enalaprilat standard solved in mobile phase is presented by the black line without any split peak (base line is nudged to prevent overlap).

Mentions: First extraction attempts were undertaken by utilizing Oasis MCX. This polymeric material is characterized by a strong cation exchanger (on sulfonic acid base) binding the carboxylic acids groups of the analytes of interest. However, purified samples showed a split peak for the selected transition of enalaprilat if determined by HPLC-MS/MS. The corresponding chromatograms revealed a peak occurring at an earlier retention time plus a second peak at the expected retention time of the compound (Figure 3).


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)

Determined split peak in serum with the transition 349.1 → 206.1 m/z during method development. The split peak was measured on several HPLC columns after SPE purification by Oasis MCX. In grey, the ion count of a low enalaprilat concentration in serum is shown that clearly identifies the split peak. As reference, the enalaprilat standard solved in mobile phase is presented by the black line without any split peak (base line is nudged to prevent overlap).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Determined split peak in serum with the transition 349.1 → 206.1 m/z during method development. The split peak was measured on several HPLC columns after SPE purification by Oasis MCX. In grey, the ion count of a low enalaprilat concentration in serum is shown that clearly identifies the split peak. As reference, the enalaprilat standard solved in mobile phase is presented by the black line without any split peak (base line is nudged to prevent overlap).
Mentions: First extraction attempts were undertaken by utilizing Oasis MCX. This polymeric material is characterized by a strong cation exchanger (on sulfonic acid base) binding the carboxylic acids groups of the analytes of interest. However, purified samples showed a split peak for the selected transition of enalaprilat if determined by HPLC-MS/MS. The corresponding chromatograms revealed a peak occurring at an earlier retention time plus a second peak at the expected retention time of the compound (Figure 3).

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