Limits...
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

Multiple reaction monitoring scan chromatograms of enalapril, enalaprilat, and benazepril. The left graph illustrates the shift in retention time detected by comparing the analytes solved in mobile phase versus the analytes in purified matrix during first extraction attempts. Right graph shows the final chromatogram by comparing also the analytes in mobile phase versus analytes in purified serum. A timely shift in retention time was not detectable anymore. MRMs: 377.2 → 234.2 m/z (enalapril), 349.1 → 206.1 m/z (enalaprilat), and 425.3 → 351.2 m/z (benazepril). The black line identifies the extracted serum samples and the grey line illustrates the drug substances dissolved in mobile phase. Cps: counts.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4383429&req=5

fig5: Multiple reaction monitoring scan chromatograms of enalapril, enalaprilat, and benazepril. The left graph illustrates the shift in retention time detected by comparing the analytes solved in mobile phase versus the analytes in purified matrix during first extraction attempts. Right graph shows the final chromatogram by comparing also the analytes in mobile phase versus analytes in purified serum. A timely shift in retention time was not detectable anymore. MRMs: 377.2 → 234.2 m/z (enalapril), 349.1 → 206.1 m/z (enalaprilat), and 425.3 → 351.2 m/z (benazepril). The black line identifies the extracted serum samples and the grey line illustrates the drug substances dissolved in mobile phase. Cps: counts.

Mentions: At an early development stage of the extraction protocol in serum at which the extraction was not methodologically sound, we encountered some deviation in retention time when comparing the chromatograms of analyte solved in mobile phase to extracted analyte in serum (Figure 5). By increasing the amount of organic washing steps as well as the elution force of the used organic solvents (methanol, acetone, and ethyl acetate), this timely shift could be eliminated (Figure 5). Even the intensities and peak areas of both, the analyte in mobile phase and the analyte in purified serum, were finally comparable. This pointed out the gained high degree of sample purification and accounted for a very limited signal suppression by the residual matrix compound in the final extract. However, it illustrates that matrix effects not only affect the signal intensities of the mass spectrometer but can also alter the chromatographic performance. A comparable effect was recently published by Fang et al. [8].


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)

Multiple reaction monitoring scan chromatograms of enalapril, enalaprilat, and benazepril. The left graph illustrates the shift in retention time detected by comparing the analytes solved in mobile phase versus the analytes in purified matrix during first extraction attempts. Right graph shows the final chromatogram by comparing also the analytes in mobile phase versus analytes in purified serum. A timely shift in retention time was not detectable anymore. MRMs: 377.2 → 234.2 m/z (enalapril), 349.1 → 206.1 m/z (enalaprilat), and 425.3 → 351.2 m/z (benazepril). The black line identifies the extracted serum samples and the grey line illustrates the drug substances dissolved in mobile phase. Cps: counts.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Multiple reaction monitoring scan chromatograms of enalapril, enalaprilat, and benazepril. The left graph illustrates the shift in retention time detected by comparing the analytes solved in mobile phase versus the analytes in purified matrix during first extraction attempts. Right graph shows the final chromatogram by comparing also the analytes in mobile phase versus analytes in purified serum. A timely shift in retention time was not detectable anymore. MRMs: 377.2 → 234.2 m/z (enalapril), 349.1 → 206.1 m/z (enalaprilat), and 425.3 → 351.2 m/z (benazepril). The black line identifies the extracted serum samples and the grey line illustrates the drug substances dissolved in mobile phase. Cps: counts.
Mentions: At an early development stage of the extraction protocol in serum at which the extraction was not methodologically sound, we encountered some deviation in retention time when comparing the chromatograms of analyte solved in mobile phase to extracted analyte in serum (Figure 5). By increasing the amount of organic washing steps as well as the elution force of the used organic solvents (methanol, acetone, and ethyl acetate), this timely shift could be eliminated (Figure 5). Even the intensities and peak areas of both, the analyte in mobile phase and the analyte in purified serum, were finally comparable. This pointed out the gained high degree of sample purification and accounted for a very limited signal suppression by the residual matrix compound in the final extract. However, it illustrates that matrix effects not only affect the signal intensities of the mass spectrometer but can also alter the chromatographic performance. A comparable effect was recently published by Fang et al. [8].

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