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

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.

Figure 1

Scale-up process and optimization steps to establish a high-throughput approach of all bioanalytical assays utilizing solid-phase extraction. On the left side the development approach of sample preparation and purification is illustrated (a). The solid-phase extraction is performed by cartridges using the vacuum manifold. By contrast the scale-up is shown on the right side (b). Samples were prepared in 96-well approaches utilizing multichannel pipettes. The purification is conducted on a positive pressure manifold with 96-well plates. For the drying process the applied thermomixer was modified by a heatable water bath and a special drying top frame to deal with the deep-well collection plates (in-house development).

Figure 2

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.

Figure 3

Determined split peak in serum with the transition 349.1 → 206.1 m/z during method development. The split peak was measured on several HPLC columns after SPE purification by Oasis MCX. In grey, the ion count of a low enalaprilat concentration in serum is shown that clearly identifies the split peak. As reference, the enalaprilat standard solved in mobile phase is presented by the black line without any split peak (base line is nudged to prevent overlap).

Figure 4

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).

Figure 5

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.

Figure 6

Effect of elution volume on peak area of the analytes of interest. The peak areas of enalapril (a), enalaprilat (b), and benazepril (c) after elution are presented with different volumes of 2% formic acid in methanol. Each determination was conducted by three independently prepared quality control samples. The mean and corresponding standard deviations are shown.

Figure 7

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.

Figure 8

The plots show the accuracy results of 22 serum calibration curves and the accuracy results of 7 urinary calibration curves (each covering 11 concentration levels per drug substance) of enalapril (black) and enalaprilat (grey) used for the evaluation of the obtained results of the conducted Phase I study. Additionally the accuracy thresholds (dashed lines) according to FDA and EMA bioanalytical guidelines for all concentrations levels (±15%) and the LLOQ (±20%) are indicated.