Author Contributions
Methodology, M.C. and R.B.; validation, M.C., R.B. and B.G.; formal analysis, M.C., R.B., B.G., M.G., P.G.C. and F.P.; investigation, M.C., R.B., B.G. and M.G.; data curation, M.C., R.B., B.G., M.G., P.G.C. and F.P.; writing—original draft preparation, M.C. and R.B.; writing—review and editing, M.C., M.G., P.G.C. and F.P.; supervision, P.V. and F.P. All authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
P.G.C. has participated in the speaker bureau for Angelini, Shionogi, Pfizer, and MSD outside of the submitted work. M.G. has participated in the speaker bureau for Angelini and Shionogi outside of the submitted work. F.P. has participated in speaker’s bureau for Angelini, BeiGene, Gilead, Menarini, Merck Sharp & Dohme, Nordic Pharma, Pfizer, and Sanofi Aventis, and in an advisory board for Advanz Pharma, Angelini, BeiGene, Gilead, Merck Sharp & Dohme, Nordic Pharma, and Pfizer, outside the submitted work. P.V. has participated in speaker bureau for Correvio, Gilead, Merck Sharp, and Dohme, Nordic Pharma, and Pfizer, outside the submitted work and in advisory board for bioMérieux, Gilead, Merck Sharp, and Dohme, Nabriva, Nordic Pharma, Pfizer, Thermo-Fisher, and Venatorx, outside the submitted work. The other authors report no potential conflicts of interest for this work.
Figure 1.
Overlayed MRM chromatograms for MRP (black line) and MRP-d6 (red line) obtained in the analysis of (a), a blank sample extracted with the methanol-IS solution, showing the absence of peaks related to MRP and the presence of a well-defined peak for MRP-d6; (b) an LLOQ sample with printed S/N ratio (SNR); (c) a real patient sample showing good peak shape and resolution of specific peaks.
Figure 1.
Overlayed MRM chromatograms for MRP (black line) and MRP-d6 (red line) obtained in the analysis of (a), a blank sample extracted with the methanol-IS solution, showing the absence of peaks related to MRP and the presence of a well-defined peak for MRP-d6; (b) an LLOQ sample with printed S/N ratio (SNR); (c) a real patient sample showing good peak shape and resolution of specific peaks.
Figure 2.
Overlayed MRM chromatograms for VBR (blue line) and AVI-C13 (green line) obtained in the analysis of (a), a blank sample extracted with the methanol-IS solution, showing the absence of peaks related to MRP and the presence of a well-defined peak of AVI-C13; (b) an LLOQ sample with printed S/N ratio (SNR); (c) a real patient sample showing good peak shape and resolution of specific peaks.
Figure 2.
Overlayed MRM chromatograms for VBR (blue line) and AVI-C13 (green line) obtained in the analysis of (a), a blank sample extracted with the methanol-IS solution, showing the absence of peaks related to MRP and the presence of a well-defined peak of AVI-C13; (b) an LLOQ sample with printed S/N ratio (SNR); (c) a real patient sample showing good peak shape and resolution of specific peaks.
Figure 3.
Example of a calibration obtained by plotting the MRP/MRM-d6 area ratio (response) over-concentration, in the 0.1–100.0 mg/L range, and software fitting of 6 experimental calibration points with the linear equation and correlation coefficient reported in the upper left corner of the box.
Figure 3.
Example of a calibration obtained by plotting the MRP/MRM-d6 area ratio (response) over-concentration, in the 0.1–100.0 mg/L range, and software fitting of 6 experimental calibration points with the linear equation and correlation coefficient reported in the upper left corner of the box.
Figure 4.
Example of calibrations obtained by plotting the VBR/AVI-C13 area ratio (response) over-concentration, in the 0.1–100.0 mg/L range, by software fitting of 6 experimental calibration points with the linear equation and correlation coefficient reported in the upper left corner of the box.
Figure 4.
Example of calibrations obtained by plotting the VBR/AVI-C13 area ratio (response) over-concentration, in the 0.1–100.0 mg/L range, by software fitting of 6 experimental calibration points with the linear equation and correlation coefficient reported in the upper left corner of the box.
Figure 5.
Box plot performed with the free online BoxPlotR tool, showing the spread of the MRP concentration measured in 42 real patients’ plasma microsamples. Population size: 42; Median: 16.75; Mean: 22.89; Minimum: 2.2; Maximum: 92.0; First quartile: 8.0; Third quartile: 29.7.
Figure 5.
Box plot performed with the free online BoxPlotR tool, showing the spread of the MRP concentration measured in 42 real patients’ plasma microsamples. Population size: 42; Median: 16.75; Mean: 22.89; Minimum: 2.2; Maximum: 92.0; First quartile: 8.0; Third quartile: 29.7.
Figure 6.
Box plot performed with the free online BoxPlotR tool, showing the spread of the VBR concentration measured in 42 real patients’ plasma microsamples. Population size: 42; Median: 24.95; Mean: 34.39; Minimum: 4.2; Maximum: 98.4; First quartile: 17.3; Third quartile: 42.4.
Figure 6.
Box plot performed with the free online BoxPlotR tool, showing the spread of the VBR concentration measured in 42 real patients’ plasma microsamples. Population size: 42; Median: 24.95; Mean: 34.39; Minimum: 4.2; Maximum: 98.4; First quartile: 17.3; Third quartile: 42.4.
Figure 7.
Chemical structure of the molecules involved in this study: VBR (upper left), MRP (upper right), the internal standards AVI-C13 (lower left), and MRP-d6 (lower right) employed for analysis.
Figure 7.
Chemical structure of the molecules involved in this study: VBR (upper left), MRP (upper right), the internal standards AVI-C13 (lower left), and MRP-d6 (lower right) employed for analysis.
Table 1.
Specific Multiple Reaction Monitoring (MRM) transition parameters used for MRP, MRP-d6 (IS), VBR, AVI-C13 (IS) acquisition.
Table 1.
Specific Multiple Reaction Monitoring (MRM) transition parameters used for MRP, MRP-d6 (IS), VBR, AVI-C13 (IS) acquisition.
Analyte | Retention Time (min) | Precursor Ion (m/z) | Production (m/z) | Dwell Time (ms) | Fragmentator (V) | Collision Energy (V) |
---|
MRP | 1.21 | 384.2 | 141.0 | 20 | 166 | 16 |
MRP-d6 | 1.20 | 390.2 | 147.1 | 20 | 166 | 16 |
VBR | 2.35 | 296.0 | 234.1 | 20 | 166 | 20 |
AVI-C13 | 1.16 | 269.0 | 96.0 | 20 | 166 | 29 |
Table 2.
Binary pump program used for linear gradient elution with mobile phases A and B.
Table 2.
Binary pump program used for linear gradient elution with mobile phases A and B.
Time (min) | A (%) | B (%) | Flow (mL/min) |
---|
0 | 95 | 5 | 0.5 |
3 | 5 | 95 | 0.5 |
3.5 | 5 | 95 | 0.5 |
3.51 | 95 | 5 | 0.5 |
4 | 95 | 5 | 0.5 |
Table 3.
Intra-day and inter-day average (avg) precision and accuracy assessed at four concentration levels (LLOQ, LQC, MQC and HQC) five times (intra-day) in three different analytical runs (inter-day) for MRP.
Table 3.
Intra-day and inter-day average (avg) precision and accuracy assessed at four concentration levels (LLOQ, LQC, MQC and HQC) five times (intra-day) in three different analytical runs (inter-day) for MRP.
QC Levels | Nominal Conc. (mg/L) | Intraday (n = 5) | Inter-Day (n = 3) |
---|
Avg Conc. (mg/L) | Avg Precision (CV%) | Avg Accuracy (Bias%) | Avg Conc. (>mg/L) | Avg Precision (CV%) | Avg Accuracy (Bias%) |
---|
LLOQ | 0.1 | 0.06 | 14.5 | 10.8 | 0.06 | 15.9 | 19.2 |
LQC | 0.25 | 0.27 | 10.5 | 9.5 | 0.28 | 10.6 | 10.3 |
MQC | 25 | 23.8 | 10.1 | 9.9 | 25.4 | 9.8 | 6.7 |
HQC | 75 | 76.9 | 9.8 | 8.7 | 73.9 | 10.2 | 4.1 |
Table 4.
Intra-day and inter-day average (avg) precision and accuracy assessed at four concentration levels (LLOQ, LQC, MQC and HQC) five times (intra-day) in three different analytical runs (inter-day) for VBR.
Table 4.
Intra-day and inter-day average (avg) precision and accuracy assessed at four concentration levels (LLOQ, LQC, MQC and HQC) five times (intra-day) in three different analytical runs (inter-day) for VBR.
QC Levels | Nominal Conc. (mg/L) | Intraday (n = 5) | Inter-Day (n = 3) |
---|
Avg Conc. (mg/L) | Avg Precision (CV%) | Avg Accuracy (Bias%) | Avg Conc. (mg/L) | Avg Precision (CV%) | Avg Accuracy (Bias%) |
---|
LLOQ | 0.1 | 0.04 | 17.5 | 19.8 | 0.04 | 17.9 | 19.1 |
LQC | 0.25 | 0.24 | 12.5 | 9.5 | 0.23 | 10.4 | 13.3 |
MQC | 25 | 26.8 | 10.9 | 9.9 | 26.4 | 9.5 | 9.7 |
HQC | 75 | 77.5 | 10.8 | 7.5 | 76.9 | 8.2 | 7.1 |
Table 5.
Average (Avg) Matrix Effect (ME%) and Extraction Recovery (ER%) of MRP measured at different concentration levels for MRP.
Table 5.
Average (Avg) Matrix Effect (ME%) and Extraction Recovery (ER%) of MRP measured at different concentration levels for MRP.
Quality Control Level | N° | Avg Me (%) | Avg IS-Normalized Me (%) | Avg ER (%) |
---|
LQC | 30 | 121.8 | 102.2 | 86.3 |
MQC | 30 | 115.5 | 104.1 | 88.5 |
HQC | 30 | 117.2 | 100.3 | 91.4 |
Table 6.
Average (Avg) Matrix effect (ME%) and Recovery (ER%) of DBV measured at different concentration levels for VBR.
Table 6.
Average (Avg) Matrix effect (ME%) and Recovery (ER%) of DBV measured at different concentration levels for VBR.
Quality Control Level | N° | Avg Me (%) | Avg IS-Normalized Me (%) | Avg ER (%) |
---|
LQC | 30 | 181.9 | 104.2 | 76.3 |
MQC | 30 | 185.7 | 105.1 | 83.5 |
HQC | 30 | 187.2 | 98.3 | 87.4 |
Table 7.
Stability of MRP at different storage conditions. In our study, we tested both the extracts and the plasma samples (according to our routine needs).
Table 7.
Stability of MRP at different storage conditions. In our study, we tested both the extracts and the plasma samples (according to our routine needs).
Quality Control | LQC | MQC | HQC |
---|
Types of Sample | Tested Conditions | Avg Accuracy (Bias%) | Avg Accuracy (Bias%) | Avg Accuracy (Bias%) |
---|
extracts | autosampler post 2 h | −20.1 | −19.5 | −24.2 |
freezer post 24 h | −19.5 | −19.7 | −21.8 |
plasma samples | freeze-thaw stability |
1 cycle | −15.2 | −15.6 | −15.8 |
2 cycle | −35.6 | −29.2 | −22.5 |
3 cycle | −67.1 | −65.2 | −56.1 |
Table 8.
Stability of VBR at different storage conditions. In our study, we tested both the extracts and the plasma samples (according to our routine needs).
Table 8.
Stability of VBR at different storage conditions. In our study, we tested both the extracts and the plasma samples (according to our routine needs).
Quality Control | LQC | MQC | HQC |
---|
Types of Sample | Tested Conditions | Avg Accuracy (Bias%) | Avg Accuracy (Bias%) | Avg Accuracy (Bias%) |
---|
extracts | autosampler post 2 h | −12.1 | −19.2 | −14.6 |
freezer post 24 h | −9.5 | −8.7 | −9.1 |
plasma samples | freeze-thaw stability |
1 cycle | −8.8 | −9.1 | −9.4 |
2 cycle | −15.1 | −19.2 | −12.6 |
3 cycle | −27.4 | −25.1 | −26.3 |