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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

Comparison of mixing ratios of serum and water on resulting peak areas. The detected peak areas of enalapril (a) and enalaprilat (b) of purified serum samples are presented. The mixing ratio was varied between 1 : 1 and 1 : 23. Each determination was conducted by three independently prepared quality control samples. The mean and corresponding standard deviations are shown.
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fig2: Comparison of mixing ratios of serum and water on resulting peak areas. The detected peak areas of enalapril (a) and enalaprilat (b) of purified serum samples are presented. The mixing ratio was varied between 1 : 1 and 1 : 23. Each determination was conducted by three independently prepared quality control samples. The mean and corresponding standard deviations are shown.

Mentions: Preliminary investigations on the degree of sample dilution had been shown to influence the extraction performance and accounted highly for a robust method with high recovery. Investigations on suitable sample dilution solvents (formic acid, phosphoric acid, hydrochloric acid, and water) and their mixing ratio with the sample itself were conducted. To determine the best suitable mixing ratio of acids or pure water, the ratio was varied between 1 : 1 and 1 : 23. The conducted investigations on the most appropriate dilution solvent showed that water is sufficient if high dilution factors were applied. By increasing the mixing ratio, the detected peak areas of enalapril and enalaprilat increased in parallel (Figure 2). A mixing ratio of 1 : 10 and 1 : 23 worked best with regard to sample recovery. The highest dilution ratio resulted in a total sample volume of about 1.2 mL. Owing to the maximum capacity of a cavity (~1.4 mL), a higher degree of dilution is not recommended for routine. It increases the risk of carryover and rises the likelihood of sample mix-up as the sample solution needs to be pipetted at least in two parts into the cavity. The final composition of the diluted sample solution consisted of 50 μL serum being mixed with 5 μL benazepril working solution (IS) and 1100 μL water.


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 mixing ratios of serum and water on resulting peak areas. The detected peak areas of enalapril (a) and enalaprilat (b) of purified serum samples are presented. The mixing ratio was varied between 1 : 1 and 1 : 23. Each determination was conducted by three independently prepared quality control samples. The mean and corresponding standard deviations are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Comparison of mixing ratios of serum and water on resulting peak areas. The detected peak areas of enalapril (a) and enalaprilat (b) of purified serum samples are presented. The mixing ratio was varied between 1 : 1 and 1 : 23. Each determination was conducted by three independently prepared quality control samples. The mean and corresponding standard deviations are shown.
Mentions: Preliminary investigations on the degree of sample dilution had been shown to influence the extraction performance and accounted highly for a robust method with high recovery. Investigations on suitable sample dilution solvents (formic acid, phosphoric acid, hydrochloric acid, and water) and their mixing ratio with the sample itself were conducted. To determine the best suitable mixing ratio of acids or pure water, the ratio was varied between 1 : 1 and 1 : 23. The conducted investigations on the most appropriate dilution solvent showed that water is sufficient if high dilution factors were applied. By increasing the mixing ratio, the detected peak areas of enalapril and enalaprilat increased in parallel (Figure 2). A mixing ratio of 1 : 10 and 1 : 23 worked best with regard to sample recovery. The highest dilution ratio resulted in a total sample volume of about 1.2 mL. Owing to the maximum capacity of a cavity (~1.4 mL), a higher degree of dilution is not recommended for routine. It increases the risk of carryover and rises the likelihood of sample mix-up as the sample solution needs to be pipetted at least in two parts into the cavity. The final composition of the diluted sample solution consisted of 50 μL serum being mixed with 5 μL benazepril working solution (IS) and 1100 μL water.

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