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

Comparison of required time for bioanalysis between development scale by applying single cartridges and the high-throughput approach with 96-well plate. The calculation bases on a sample amount of 96 samples. The black areas mark the required time for sample purification by solid-phase extraction and grey areas identify the time frame required for sample preparation. The dashed line represents one full working day (8 hours). By applying the high-throughput approach the sample preparation and purification is finalized within 2 hours while the same amount of samples is impossible to purify within one working day by one lab technician using the previous development scale.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4383429&req=5

fig7: Comparison of required time for bioanalysis between development scale by applying single cartridges and the high-throughput approach with 96-well plate. The calculation bases on a sample amount of 96 samples. The black areas mark the required time for sample purification by solid-phase extraction and grey areas identify the time frame required for sample preparation. The dashed line represents one full working day (8 hours). By applying the high-throughput approach the sample preparation and purification is finalized within 2 hours while the same amount of samples is impossible to purify within one working day by one lab technician using the previous development scale.

Mentions: The transfer from vacuum extraction to positive pressure not only enabled a semiautomated extraction but also highly increased the sample amount up to about thousand samples which can be purified per week by one laboratory technician. Since appropriate equipment for rapid and continuous drying was not commercially available, required equipment for the drying process was self-developed to suit best the laboratory preconditions and needs. Figure 1 is enclosed illustrating the scale-up from single cartridges using vacuum technique to the positive pressure extraction in 96-well formate. As indicated in Figure 7, the scale-up is a mandatory step in method development if the assay will be applied to analyze hundreds or thousands of samples. In our positive pressure approach, it took about 2 hours from raw sample to sample preparation and extraction to the final sample solution ready to be determined by HPLC-MS/MS. In such a run, 96 samples could be prepared which required much more than one full working day (8 h) of one laboratory technician to prepare the same amount by the previously used vacuum manifold approach. The applied pressures of 1–3 psi were fully sufficient to ensure a continuous and appropriate flow rate of solvent/sample solution through the sorbent material. The scale-up enabled a nearly sixfold higher sample throughput.


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)

Comparison of required time for bioanalysis between development scale by applying single cartridges and the high-throughput approach with 96-well plate. The calculation bases on a sample amount of 96 samples. The black areas mark the required time for sample purification by solid-phase extraction and grey areas identify the time frame required for sample preparation. The dashed line represents one full working day (8 hours). By applying the high-throughput approach the sample preparation and purification is finalized within 2 hours while the same amount of samples is impossible to purify within one working day by one lab technician using the previous development scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Comparison of required time for bioanalysis between development scale by applying single cartridges and the high-throughput approach with 96-well plate. The calculation bases on a sample amount of 96 samples. The black areas mark the required time for sample purification by solid-phase extraction and grey areas identify the time frame required for sample preparation. The dashed line represents one full working day (8 hours). By applying the high-throughput approach the sample preparation and purification is finalized within 2 hours while the same amount of samples is impossible to purify within one working day by one lab technician using the previous development scale.
Mentions: The transfer from vacuum extraction to positive pressure not only enabled a semiautomated extraction but also highly increased the sample amount up to about thousand samples which can be purified per week by one laboratory technician. Since appropriate equipment for rapid and continuous drying was not commercially available, required equipment for the drying process was self-developed to suit best the laboratory preconditions and needs. Figure 1 is enclosed illustrating the scale-up from single cartridges using vacuum technique to the positive pressure extraction in 96-well formate. As indicated in Figure 7, the scale-up is a mandatory step in method development if the assay will be applied to analyze hundreds or thousands of samples. In our positive pressure approach, it took about 2 hours from raw sample to sample preparation and extraction to the final sample solution ready to be determined by HPLC-MS/MS. In such a run, 96 samples could be prepared which required much more than one full working day (8 h) of one laboratory technician to prepare the same amount by the previously used vacuum manifold approach. The applied pressures of 1–3 psi were fully sufficient to ensure a continuous and appropriate flow rate of solvent/sample solution through the sorbent material. The scale-up enabled a nearly sixfold higher sample throughput.

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