Correction to: Fit-for-Purpose Quality Control System in Continuous Bioanalysis during Long-Term Pediatric Studies.

The LENA collaborator list below was not included in the original article.s.

A quality control system for ligand-binding assay of plasma renin activity: Proof-of-concept within a pharmacodynamic study.

While the role of plasma renin activity (PRA) in heart failure has been widely studied in adults, comprehensive data on pediatric heart failure remain lacking. This drawback is increasingly being addressed by academic research. Nevertheless, such pediatric investigations are commonly conducted only once due to ethical constraints. Therefore, the quality of bioanalytical data must be ensured to acquire meaningful insights into maturing humoral parameters. However, appropriate post-validation assessment of bioanalytical runs is currently underrepresented by regulatory guidance. Thus, for applications in an academic environment, an easy-to-handle six-step bioanalytical quality control system was designed based on regulatory guidelines (e.g. U.S. Food and Drug Administration) combined with international recommendations (e.g. Clinical and Laboratory Standards Institute) and current scientific discussion. Its applicability to an enzyme-linked immunosorbent assay for determination of PRA was investigated within three pediatric trials of the EU-funded "Labeling of Enalapril in Neonates up to Adolescents" project. This quality control system identified 15 % bioanalytical runs as non-compliant to the predefined specifications and ensured the reliable quantification of 940 pharmacodynamic samples. The inter-run assessment of quality controls was able to demonstrate the comparability of the study results. Furthermore, 86 % of incurred sample reanalysis pairs complied with regulatory requirements (>67 %), thus underlining the long-term reproducibility of the utilized ligand-binding assay. Successful participation in interlaboratory testing confirmed the accuracy of the applied method throughout the entire study period. Further investigations showed no notable differences between the five applied lots of the PRA assay. The applicability of this quality control system was proven in an academic environment and ensured reliable results for PRA over the entire 24-month study period.

Fit-for-Purpose Quality Control System in Continuous Bioanalysis During Long-Term Pediatric Studies.

Pharmacokinetic studies are key to evidence-based pharmacotherapy. The reliability of pharmacokinetic parameters is closely related to the quality of bioanalytical data. Bioanalytical method validation is fully described by regulatory guidelines; however, it is conducted just once. To ensure reliability and comparability of clinical data, appropriate quality control systems must be enforced to monitor post-validation bioanalytical runs. While single bioanalytical run evaluation is described in international guidelines, somehow, the long-term reproducibility of the bioanalytical method is unattended; it becomes pivotal with the involvement of pediatric population. Therefore, a customized quality control system was developed that addresses regulatory requirements and encompasses the specific demands of pediatric research. It consisted of continuous multi-parameter assessment, including calibration curves, quality control samples, incurred sample reanalysis, and internal standard data. The recommendations provided by the guidelines were combined with the additional Westgard rules, statistical evaluation, and graphical observations. The applicability of the developed quality control system was investigated by using data from three pediatric clinical trials, where the system was able to identify 16% of all analytical runs as invalid. Using a pooled standard deviation provided a better estimate of long-term reproducibility by calculating the %CV, which ranged from 3.6 to 10.3% at all quality control levels. Irrespective of the difficulties encountered owing to vulnerable pediatric populations, the incurred sample reanalysis fulfilled the regulatory requirement of at least 67%. This quality control approach ensured reliable and comparable results over a whole 31-month duration in relation to pediatric studies.

Tailored Assays for Pharmacokinetic and Pharmacodynamic Investigations of Aliskiren and Enalapril in Children: An Application in Serum, Urine, and Saliva.

OBJECTIVES: Drugs that are effectively used to treat hypertension in adults (e.g., enalapril) have not been sufficiently investigated in children. Studies required for pediatric approval require special consideration regarding ethics, study design, and conduct and are also associated with special demands for the bioanalytic method. Pediatric-appropriate assays can overcome these burdens and enable systematic investigations of pharmacokinetics and pharmacodynamic in all pediatric age groups. METHODS: Tailored assays were developed for pharmacokinetic investigation of a drug in 100 µL of serum, saliva, and urine. All assays were applied in a proof-of-concept study to 22 healthy volunteers who had been given 300 mg aliskiren hemifumarate or 20 mg enalapril maleate and allowed for dense sampling. Changes in humoral parameters of the renin-angiotensin-aldosterone system were also evaluated with 6 parameters in 2.1 mL blood per time point. RESULTS: The pharmacokinetic results of aliskiren and enalapril obtained by low-volume assays in serum and urine were comparable to that noted in the literature. The dense sampling enabled very detailed concentration-time profiles that showed high intersubject variability and biphasic absorption behavior of aliskiren. The replacement of invasive sampling by saliva collection appears inappropriate for both drugs because the correlations of drug concentrations in both fluids were low. A low-volume assay was also used to determine values for in the renin-angiotensin-aldosterone system and to compare those results with the published literature. CONCLUSION: These results support both the use of low-volume assays in pediatric research and the systematic investigation of their use in neonates and infants. Use of this assay methodology will increase information about drug pharmacokinetics and pharmacodynamics in this vulnerable population and might contribute to safe and effective use of pharmacotherapy.

Simultaneous quantitative and qualitative analysis of aliskiren, enalapril and its active metabolite enalaprilat in undiluted human urine utilizing LC-ESI-MS/MS.

The benefit-risk ratio of combined blocking by the direct renin inhibitor aliskiren and an angiotensin-converting enzyme inhibitor (e.g. enalapril) on the renin-angiotensin-aldosterone system is discussed. No method was available for simultaneous determination of both drugs in urine. A novel sensitive method for simultaneous quantification in undiluted human urine was developed which enables systematic pharmacokinetic investigations, especially in poorly investigated populations like children. Matrix effects were clearly reduced by applying solid-phase extraction followed by a chromatographic separation on Xselect(TM) C18 CSH columns. Mobile phase consisted of methanol and water, both acidified with formic acid. Under gradient conditions and a flow rate of 0.4 mL/min the column effluent was monitored by tandem mass spectrometry with electrospray ionization. Calibration curves were constructed in the range of 9.4-9600 ng/mL regarding aliskiren, 11.6-12000 ng/mL for enalapril and 8.8-9000 ng/mL for enalaprilat. All curves were analyzed utilizing 1/x(2) -weighted quadratic squared regression. Intra-run and inter-run precision were 3.2-5.8% and 6.1-10.3% for aliskiren, 2.4-6.1% and 3.9-7.9% for enalapril as well as 3.1-9.4% and 4.7-12.7% regarding enalaprilat. Selectivity, accuracy and stability results comply with current international bioanalysis guidelines. The fully validated method was successfully applied to a pharmacokinetic investigation in healthy volunteers.

Liquid chromatography-tandem mass spectrometry method for determination of aliskiren in saliva and its application to a clinical trial with healthy volunteers.

Although serum and plasma are the biological fluids of choice for pharmacokinetic determination of drugs in adults, it is desirable to elucidate noninvasive methods which can be used for investigations in vulnerable groups such as children. If the drug properties grant sufficient penetration of the drug from blood into saliva, the latter is a useful matrix for noninvasive investigations. Concerning the known physicochemical properties, the direct renin inhibitor aliskiren is one of the substances of which saliva concentrations could substitute blood concentrations for pharmacokinetic investigations in children. Therefore, a reliable bioanalytical method was successfully developed and validated according to the criteria of current international bioanalytical guidelines to enable the comparison of blood and saliva concentrations of aliskiren. After purification of the fluid by solid-phase extraction the chromatographic separation was conducted by using Xselect™ C18 CSH columns. Applying a mobile phase gradient of acidified methanol and acidified water at a flow rate of 0.4ml/min the column effluent was monitored during a total run time of 7.5min by tandem mass spectrometry with electrospray ionization. Running in positive mode the following transitions were investigated: 552.2-436.2m/z for aliskiren and 425.3-351.2m/z for benazepril (internal standard). Calibration curves were constructed in the range of 0.586-1200ng/ml and were analyzed utilizing 1/x(2) weighted linear regression. Intra-run and inter-run precision were 3.8-8.1% and 3.4-8.9%. The method provides selectivity, linearity and accuracy. The validated method was then applied to determine aliskiren concentrations in saliva and blood of three healthy volunteers after oral administration of 300mg aliskiren.

Determination of aliskiren in human serum quantities by HPLC-tandem mass spectrometry appropriate for pediatric trials.

The orally active direct renin inhibitor aliskiren is approved for the treatment of essential hypertension in adults. Analytical methods utilized in clinical studies on efficacy and safety have not been fully described in the literature but need a large sample volume ranging from 200 to 700 µL, rendering them unsuitable particularly for pediatric applications. In the assay presented only 100 µL of serum is needed for mixed-mode solid-phase extraction. The chromatographic separation was performed on Xselect(TM) C18 CSH columns with mobile phase consisting of methanol-water-formic acid (75:25:0.005, v/v/v) and a flow rate of 0.4 mL/min. Running in positive electrospray ionization and multiple reaction monitoring the mass spectrometer was set to analyze precursor ion 552.2 m/z [M + H](+) to product ion 436.2 m/z during a total run time of 5 min. The method covers a linear calibration range of 0.146-1200 ng/mL. Intra-run and inter-run precisions were 0.4-7.2 and 0.6-12.9%. Mean recovery was at least 89%. Selectivity, accuracy and stability results comply with current European Medicines Agency and Food and Drug Administration guidelines. This successfully validated LC-MS/MS method with a wide linear calibration range requiring small serum amounts is suitable for pharmacokinetic investigations of aliskiren in pediatrics, adults and the elderly.