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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 different extraction methods on the internal standard normalized matrix effect. The SPE extraction by Oasis MCX is compared to the two-step extraction by Oasis WAX + MCX. Each boxplot describes median, 25th, 75th percentile + 10th, and 90th percentile as whisker. N = 9 measurements per boxplot. Statistical analysis was performed by a Mann-Whitney-U test (two-tailed P value).
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fig4: Comparison of different extraction methods on the internal standard normalized matrix effect. The SPE extraction by Oasis MCX is compared to the two-step extraction by Oasis WAX + MCX. Each boxplot describes median, 25th, 75th percentile + 10th, and 90th percentile as whisker. N = 9 measurements per boxplot. Statistical analysis was performed by a Mann-Whitney-U test (two-tailed P value).

Mentions: At this stage an excuse to an already established and fully validated extraction method for the same drug entities in urine (amongst others) is made to introduce another useful approach to purify the sample matrix, to reduce the relative matrix effects, and to meet the current EMA bioanalytical guideline [4]. A two-step solid-phase extraction by a weak anion exchanger followed by a strong cation exchanger significantly reduced the internal standard normalized matrix effect compared to the purification by MCX only (Figure 4). The more hydrophilic the compound was the merrier the improvement of the matrix effect was pronounced. However, it expounds how diverse the several biological fluids are and emphasizes the high required effort in method development to reduce the relative matrix effect. By applying the final two-step extraction the following results for the relative matrix effect were obtained: at the LLOQ the CV was 4.04% and 6.62% for enalapril and enalaprilat, respectively. A CV of 1.26% for enalapril and 1.25% for enalaprilat was calculated at the ULOQ and was therefore well within the EMA requirements of 15% [7]. This bioanalytical urinary method was fully validated according to the strictest validation parameters of EMA and FDA bioanalytical guidance [7].


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 different extraction methods on the internal standard normalized matrix effect. The SPE extraction by Oasis MCX is compared to the two-step extraction by Oasis WAX + MCX. Each boxplot describes median, 25th, 75th percentile + 10th, and 90th percentile as whisker. N = 9 measurements per boxplot. Statistical analysis was performed by a Mann-Whitney-U test (two-tailed P value).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Comparison of different extraction methods on the internal standard normalized matrix effect. The SPE extraction by Oasis MCX is compared to the two-step extraction by Oasis WAX + MCX. Each boxplot describes median, 25th, 75th percentile + 10th, and 90th percentile as whisker. N = 9 measurements per boxplot. Statistical analysis was performed by a Mann-Whitney-U test (two-tailed P value).
Mentions: At this stage an excuse to an already established and fully validated extraction method for the same drug entities in urine (amongst others) is made to introduce another useful approach to purify the sample matrix, to reduce the relative matrix effects, and to meet the current EMA bioanalytical guideline [4]. A two-step solid-phase extraction by a weak anion exchanger followed by a strong cation exchanger significantly reduced the internal standard normalized matrix effect compared to the purification by MCX only (Figure 4). The more hydrophilic the compound was the merrier the improvement of the matrix effect was pronounced. However, it expounds how diverse the several biological fluids are and emphasizes the high required effort in method development to reduce the relative matrix effect. By applying the final two-step extraction the following results for the relative matrix effect were obtained: at the LLOQ the CV was 4.04% and 6.62% for enalapril and enalaprilat, respectively. A CV of 1.26% for enalapril and 1.25% for enalaprilat was calculated at the ULOQ and was therefore well within the EMA requirements of 15% [7]. This bioanalytical urinary method was fully validated according to the strictest validation parameters of EMA and FDA bioanalytical guidance [7].

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