3.1. Selectivity
The selectivity of the method was assessed by examining the absence of any interfering peaks that co-eluted with tiaprofenic acid and the Internal Standard (IS) at their respective retention times. The results revealed that the interference percentage (%) for tiaprofenic acid at the Lower Limit of Quantification (LLOQ) was below 20%, while for the IS, it remained below 5%. These findings confirm the method's selectivity and are summarized in Table 1 for reference.
Table 1
Human plasma | Batch-1 | Batch − 2 | Batch − 3 | Batch − 4 | Batch − 5 | Batch − 6 |
Interference percentage (%) of tiaprofenic acid | 7.38 | 3.79 | 1.50 | 0.98 | 0.77 | 0.72 |
Interference percentage (%) of IS | 0.02 | 0.04 | 0.02 | 0.01 | 0.17 | 0.01 |
3.2. Specificity
The specificity of our method was rigorously evaluated through a meticulous examination of chromatograms representing different scenarios. Figure 1 displays these chromatograms, which include those for blank plasma, blank plasma spiked with tiaprofenic acid at the Lower Limit of Quantification (LLOQ) concentration, and blank plasma spiked with the Internal Standard (IS) at the concentration utilized in this study.
The key criterion for assessing specificity was the determination of retention times, which were found to be approximately 5 minutes for tiaprofenic acid and 7.4 minutes for IS. Significantly, the examination of these chromatograms revealed the complete absence of any interfering peaks in the blank plasma at the specific retention times corresponding to tiaprofenic acid and IS. This unequivocal absence of interferences reinforces the method's exceptional specificity, affirming its suitability for precise and accurate analytical purposes.
3.3. Matrix effect
In order to comprehensively assess the matrix effect, extraction recovery, and process efficiency of tiaprofenic acid, we present the findings in Table 2. This analysis is crucial for understanding the impact of the biological matrix on the analytical method's performance.
Table 2
| Tiaprofenic acid |
| LQC (200 ng/ml) | HQC (40000ng/ml) |
Tiprofenic acid preparing in mobile phase | Tiaprofenic Acid preparing in extracted blank plasma | Tiaprofenic acid preparing in blank plasma before extraction | Tiprofenic acid preparing in mobile phase | Tiaprofenic Acid preparing in extracted blank plasma | Tiaprofenic acid preparing in blank plasma before extraction |
Plasma control-1 | Area Mean | 18145.01 | 18703.82 | 18934.87 | 6052813.13 | 5469410.50 | 5465336.64 |
STD | 1128.68 | 193.90 | 249.21 | 844062.99 | 10848.04 | 351220.47 |
CV % | 6.22 | 1.04 | 1.32 | 13.94 | 0.20 | 6.43 |
ME | 103.08 | 90.36 |
ER | 104.35 | 90.29 |
PE | 101.24 | 99.93 |
Plasma control-2 | Area Mean | 18145.01 | 19359.94 | 18053.78 | 6052813.13 | 6465180.12 | 5689189.69 |
Standard deviation | 1128.68 | 160.03 | 250.29 | 844062.99 | 89070.50 | 118996.97 |
CV % | 6.22 | 0.83 | 1.39 | 13.94 | 1.38 | 2.09 |
ME | 106.70 | 106.81 |
ER | 99.50 | 93.99 |
PE | 93.25 | 88.00 |
Plasma control-3 | Area Mean | 18145.01 | 19658.51 | 18567.70 | 6052813.13 | 6465180.12 | 6255489.38 |
Standard deviation | 1128.68 | 173.92 | 244.13 | 844062.99 | 89070.50 | 197894.89 |
CV % | 6.22 | 0.88 | 1.31 | 13.94 | 1.38 | 3.16 |
ME | 108.34 | 106.81 |
ER | 102.33 | 103.35 |
PE | 94.45 | 96.76 |
Plasma control-4 | Area Mean | 18145.01 | 18289.31 | 17839.32 | 6052813.13 | 5469410.50 | 6368561.23 |
Standard deviation | 1128.68 | 141.66 | 251.53 | 844062.99 | 10848.04 | 129947.42 |
CV % | 6.22 | 0.77 | 1.41 | 13.94 | 0.20 | 2.04 |
ME | 100.80 | 90.36 |
ER | 98.32 | 105.22 |
PE | 97.54 | 116.44 |
Plasma control-5 | Area Mean | 18145.01 | 19907.54 | 18573.06 | 6052813.13 | 5703717.02 | 5749054.35 |
Standard deviation | 1128.68 | 113.04 | 296.31 | 844062.99 | 29775.83 | 127751.15 |
CV % | 6.22 | 0.57 | 1.60 | 13.94 | 0.52 | 2.22 |
ME | 109.71 | 94.23 |
ER | 102.36 | 94.98 |
PE | 93.30 | 100.79 |
Plasma control-6 | Area Mean | 18145.01 | 18572.06 | 19203.24 | 6052813.13 | 5693190.39 | 5023272.32 |
Standard deviation | 1128.68 | 270.37 | 210.87 | 844062.99 | 237057.05 | 132605.99 |
CV % | 6.22 | 1.46 | 1.10 | 13.94 | 4.16 | 2.64 |
ME | 102.35 | 94.06 |
ER | 105.83 | 82.99 |
PE | 103.40 | 88.23 |
Remarkably, regardless of the concentration levels tested for tiaprofenic acid, no significant matrix effect was observed. The matrix effect (ME) values fell within the range of [90% − 109%], suggesting minimal interference from the matrix in the analysis. The process efficiency (PE) remained within the range of [87% − 116%], indicating the method's effectiveness in extracting the analyte from the matrix. Furthermore, the extraction recovery (ER) values obtained for both low and high concentrations of tiaprofenic acid were between [82% − 105%], affirming the method's reliability in recovering the analyte from the biological matrix.
These findings collectively reinforce the robustness and reliability of our analytical method in the presence of diverse biological matrices, highlighting its suitability for accurately quantifying tiaprofenic acid.
3.4. Calibration and response function
Our calibration process for tiaprofenic acid meticulously evaluated various regression models to determine the most suitable model for calculating sample concentrations accurately. The assessment revealed that a quadratic model with a weighting of 1/concentration^2 provided the most robust and accurate results across the calibration curve. This model selection is crucial, as it directly impacts the accuracy and reliability of concentration calculations.
The nominal percentages of the three distinct calibration ranges are presented in Table 3. These ranges cover a wide spectrum of concentrations, ensuring the method's applicability across a broad analytical range. It is noteworthy that the accuracy of the back-calculated concentrations for each calibration standard falls comfortably within the stringent criterion of ± 15%. This indicates the method's precision and reliability in accurately quantifying tiaprofenic acid concentrations, regardless of the specific concentration levels.
Table 3
| | Nominal concentration (ng/mL) |
100 | 500 | 1000 | 5000 | 10000 | 12500 | 25000 | 50000 |
Accuracy (%) | 1 | 97.19 | 102.36 | 102.22 | 98.67 | 101.54 | 90.86 | 105.80 | 100.16 |
2 | 97.95 | 100.56 | 100.14 | 100.80 | 100.18 | 97.72 | 100.72 | 104.51 |
3 | 104.46 | 90.25 | 88.71 | 106.94 | 105.93 | 105.52 | 103.77 | 98.58 |
Table 4
accuracy and precision result
Analyte | Nominal Conc. (ng/mL) | Within-run (n = 6) | Between-run (n = 30) |
Calculated Conc. (ng/mL) Mean ± SD | Accuracy (%) | Precision | Calculated Conc. (ng/mL) Mean ± SD | Accuracy (%) | Precision |
Tiaprofenic acid | LLOQ (100 ng/mL) | 94.8 ± 2.17 | 94 | 2.28 | 96.2 ± 11.5 | 96.2 | 11.9 |
QCL(200 ng/mL) | 207 ± 19.1 | 103 | 2.24 | 197 ± 14.8 | 98.3 | 7.51 |
QCM(20000 ng/mL) | 20000 ± 900 | 100 | 4.49 | 20600 ± 1490 | 103 | 7.26 |
QCH(40000 ng/mL) | 37300 ± 2500 | 93.3 | 6.69 | 40700 ± 2190 | 102 | 5.39 |
These findings collectively validate the robustness and accuracy of our calibration process and demonstrate the method's suitability for precise quantification of tiaprofenic acid concentrations across a wide range of values. This level of accuracy is essential for various bioanalytical applications, where reliable concentration determination is paramount.
3.5. Accuracy and precision
The accuracy of our method was calculated by comparing the determined concentrations to the nominal values. Across all concentration levels, ranging from LLOQ to CQH, the accuracy consistently ranged from 80.9–109% of the nominal values. These accuracy percentages indicate that our method provides reliable and consistent measurements, as they are well within acceptable limits.
Precision, often expressed as the coefficient of variation (%CV), provides insights into the repeatability and reproducibility of the method. The precision values, expressed as %CV, ranged from 0.45–11.9%. These precision values are indicative of excellent repeatability and reproducibility, as they are substantially below the acceptable limit of 20% at the LLOQ and 15% at all other concentration levels.
The combined assessment of accuracy and precision underscores the robustness and reliability of our method for tiaprofenic acid quantification. Notably, at all concentration levels, the accuracy and precision values remained well within the predefined acceptance criteria. Specifically, accuracy values were < 20% at the LLOQ and < 15% at all other levels. Consequently, our method meets the stringent criteria for accuracy and precision, reinforcing its suitability for accurate tiaprofenic acid determination in diverse analytical applications.
These results unequivocally establish the robustness and reliability of our method for tiaprofenic acid quantification. The method's consistent accuracy and precision, well within stringent acceptance criteria, demonstrate its suitability for precise and reliable tiaprofenic acid analysis across a wide range of concentrations. This level of analytical performance is fundamental to various bioanalytical applications where accuracy and precision are paramount.
The accuracy profile, depicted in Fig. 2, plays a critical role in assessing the method's reliability and its ability to consistently produce accurate results. This assessment incorporates tolerance intervals within the acceptance limits, emphasizing a crucial aspect of method validation.
The inclusion of tolerance intervals within the acceptance limits signifies a statistical assurance that the method can reliably deliver results meeting predefined criteria. Specifically, this means that the probability of the difference between the calculated concentrations and the reference values remaining below the acceptance limit exceeds the chosen β value, set as a proportion of future results at 90%.
In practical terms, this indicates that across a concentration range spanning from 100 ng/ml to 40000 ng/ml, the analyst can have confidence that the method consistently maintains an average β probability of producing results falling within the acceptable limits. This statistical assurance provides a high level of confidence in the method's ability to yield accurate and precise results consistently over a wide range of tiaprofenic acid concentrations.
This utilization of tolerance intervals and the β value underscores the method's robustness and its suitability for bioanalytical applications where maintaining consistent accuracy and precision is essential. It offers a valuable tool for quality control and assurance, enhancing the method's reliability and trustworthiness in various analytical settings.
3.6. Stability Assessment
Ensuring the stability of tiaprofenic acid under various conditions is integral to establishing the reliability and applicability of our method. The stability tests conducted under different scenarios yielded encouraging results, reaffirming the method's robustness.
3.6.1. Freeze-Thaw Stability
Tiaprofenic acid exhibited remarkable stability during three consecutive freeze-thaw cycles. The mean calculated concentrations for both low (QCL) and high (QCH) quality controls, as shown in Table 5, remained consistent, with accuracy percentages ranging from 101.6–111.7%. This outcome underscores the method's suitability for handling samples subject to multiple freeze-thaw cycles, a condition often encountered in real-world sample processing.
Table 5
Stability experiments | Mean calculated Conc. (n = 3) | Accuracy (%) |
QCL (200 ng/mL) | QCH (40000 ng/mL) | QCL (200 ng/mL) | QCH (40000 ng/mL) |
Freeze-thaw matrix stability (3rd cycles) | 203.2 | 44668 | 101.6 | 111.7 |
Bench top (short-term) matrix stability (8 h at 22°C) | 225.5 | 42619 | 112.7 | 106.5 |
Auto-sampler matrix stability (72 h at 10°C) | 201.7 | 43463 | 100.8 | 108.6 |
Long-term matrix stability (164j at -25°C) | 229.1 | 43585 | 114.5 | 105.11 |
3.6.2. Bench Top (Short-Term) Stability
Short-term benchtop stability, conducted over 8 hours at 22°C, demonstrated the resilience of tiaprofenic acid. The calculated concentrations for QCL and QCH quality controls remained stable, with accuracy percentages of 112.7% and 106.5%, respectively. This result indicates that the method is well-suited for short-term sample storage at ambient conditions without compromising accuracy.
3.6.3. Auto-Sampler Stability
The method also exhibited commendable stability during auto-sampler storage for an extended period of 72 hours at 10°C. The calculated concentrations for both QCL and QCH quality controls remained consistent, with accuracy percentages of 100.8% and 108.6%, respectively. This finding is essential for analytical workflows that involve automated sample handling and storage.
3.6.4. Long-Term Stability
Perhaps most impressively, tiaprofenic acid demonstrated exceptional long-term stability during a 164-day storage period at -25°C. The calculated concentrations for QCL and QCH quality controls remained virtually unchanged, with accuracy percentages of 114.5% and 105.11%, respectively. This remarkable stability over an extended duration highlights the method's reliability for long-term sample storage, a critical aspect of bioanalytical studies.
3.7. Hemolysis Effect
The assessment of the hemolysis effect on tiaprofenic acid concentrations at both QCL and QCH using hemolyzed plasma produced encouraging results. The accuracy values obtained for each concentration were 97.2% and 99.7%, respectively, both well below the accepted threshold of 15%. This outcome firmly establishes the absence of a hemolysis effect, affirming the method's suitability for analyzing plasma samples that may be subject to hemolysis.
3.8. Dilution Integrity Test
The dilution integrity test, conducted on a plasma sample loaded with twice the concentration of CQH (40000 ng/mL), yielded accuracy and precision results of 96.08% and 8.21%, respectively. These results indicate that tiaprofenic acid samples can be reliably diluted by a factor of 2, extending the range of the calibration curve. This flexibility in sample dilution is valuable in situations where samples with high concentrations need to be analyzed accurately.
3.9. Auto-Sampler Carryover Test
To investigate the potential carryover effect of the auto-sampler, a sample of blank plasma was injected immediately after a sample from the upper limit of quantification of tiaprofenic acid. The interference percentages for tiaprofenic acid and IS were found to be 0.62% and 0.015%, respectively, both comfortably below the 20% threshold for tiaprofenic acid and 5% for IS. This outcome provides strong evidence of the absence of auto-sampler carryover for tiaprofenic acid and IS, ensuring the integrity of subsequent sample analyses.