Normalization Factors in LC-ESI-MS
3.3. Results and discussions
3.3.1. Theory of the PCI-IS method in combination with MNFs
The PCI-IS method has previously been demonstrated to effectively correct MEs in LC-ESI-MS [14, 15], and all of the previous studies applied the PCI-IS method to the same type of sample matrix. We observed that poor correction efficiency was obtained when the PCI-IS method was applied to quantify analytes in different biofluids that exhibited distinct MEs, such as the plasma and CSF that were investigated in this study. To overcome this problem, we modified the PCI-IS method to be able to quantify target analytes in different biofluids.
The degree of ion suppression (or ion enhancement) at each time point can be measured using the PCI-IS response changes. The observed response ratio of an analyte to the PCI-IS can be described by the following:
𝑅𝑅𝑎𝑛𝑎𝑙𝑦𝑡𝑒,𝑥
𝑃𝐶𝐼−𝐼𝑆,𝑥, = 𝐴𝑎𝑛𝑎𝑙𝑦𝑡𝑒,𝑥∗𝐶𝑎𝑛𝑎𝑙𝑦𝑡𝑒,𝑠,𝑥
𝐴𝑃𝐶𝐼−𝐼𝑆,𝑥∗𝐶𝑃𝐶𝐼−𝐼𝑆,𝑥 = 𝐹𝑥×𝐶𝑎𝑛𝑎𝑙𝑦𝑡𝑒,𝑠,𝑥
𝐶𝑃𝐶𝐼−𝐼𝑆,𝑥 (Eq. 1)
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Aanalyte,x represents the analyte’s ability to form signals at time point x and is determined by the analyte’s physicochemical properties (e.g., pKa, proton affinity,
hydrophobicity, and hydrophilicity). Aanalyte,x is influenced by the surrounding ionization conditions (e.g., mobile phase viscosity, surface tension, and nonvolatile components in the coeluent). C represents the concentration. The subscript x represents the time point.
Aanalyte,x / APCI-IS,x was simplified to Fx to show the ratio of their ionization abilities at time point x.
When the analyte is presented in different biofluids, the different materials that coelute with the analytes give different Fx values. Using biofluid 1 (M1) and biofluid 2
(M2) as an example, we can obtain Eq. 2 from Eq. 1. When CPCI-IS,x is held constant, the analyte to PCI-IS response ratios divided by Fx should be proportional to the analyte concentrations. We define FM / FSTD as the matrix normalization factor (MNFM) and used it to associate the signal response ratios
between different biofluids:
𝑅𝑎𝑛𝑎𝑙𝑦𝑡𝑒,𝑆𝑇𝐷,𝑥
𝑅𝑃𝐶𝐼−𝐼𝑆,𝑆𝑇𝐷,𝑥 = 𝑅𝑎𝑛𝑎𝑙𝑦𝑡𝑒,𝑀1,𝑥
𝑅𝑃𝐶𝐼−𝐼𝑆,𝑀1,𝑥 × 𝑀𝑁𝐹𝑀1 =𝑅𝑎𝑛𝑎𝑙𝑦𝑡𝑒,𝑀2,𝑥
𝑅𝑃𝐶𝐼−𝐼𝑆,𝑀2,𝑥 × 𝑀𝑁𝐹𝑀2 (Eq.3)
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Accordingly, the different response ratios between the analytes and the PCI-IS in the different biofluids can be corrected by MNFM1 and MNFM2. To obtain the specific MNFM1 and MNFM2 values for each biofluid, the analyte to PCI-IS ratios at each time point in the standard, M1, and M2 should be tested before quantification. Detailed descriptions of the use of MNFs and the PCI-IS method for the correction of MEs are provided in the methods section (section 3.2.4). The MNF provides a convenient method for the quantification of the same target analytes in various biofluids by simply utilizing the calibration curve generated by standards.
3.3.2. Using the PCI-IS method in combination with MNFs to quantify etoposide and
etoposide catechol in plasma and CSF
3.3.2.1. Optimization of the sample pretreatment method for the plasma and CSF
samples
The high salt and protein concentrations in biological samples often hinder chromatographic performance, and many sample pretreatment approaches have been proposed for chromatographic bioanalyses. [1, 6, 10, 15] Among these, the protein precipitation (PPT) method has the benefit of few preparation steps and was selected as the sample pretreatment method in the present study. [6, 10]
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In comparison with plasma samples, the protein contained in CSF has been rarely discussed. Most of the previous studies describe PPT for CSF prior to a chromatographic analysis, but proof that this step is necessary is rarely provided. To rigorously investigate the protein content in CSF to propose an efficient sample preparation procedure for CSF, we used the Western blot method to measure the protein content. ACN and methanol were chosen as the organic solvents for PPT. Different volumes and types of organic solvents were added to CSF and plasma for PPT, and the amount of the residual protein is displayed in Figure 3.1: Lane (a) shows the protein content of a 1,000-times-diluted human plasma sample. The plasma samples deproteinized by a 4x volume of methanol showed lower protein contents than those deproteinized by a 2x volume of ACN (Figure 3.1, Lanes (b) and (c)). All of the CSF samples with and without protein precipitation exhibited very low protein contents. The CSF sample without any preparation showed an even lower protein residue than the plasma protein-precipitated samples (Figure 3.1, Lanes (d), (e), and (f)). Although the addition of organic solvent to CSF could decrease the level of residual protein, it will also dilute the drug in the CSF sample, at the expense of the method’s sensitivity.
Because the CSF drug concentration is very low, the CSF samples were filtered and
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directly injected into the LC column. The plasma samples that were deproteinized by 4x volume of methanol were used for the LC-ESI-MS analysis.
3.3.2.2. Improvement of the quantification accuracy by using the PCI-IS method in
combination with MNFs
After establishing the sample pretreatment method, the PCI-IS method in combination with MNFs was applied to quantify etoposide and etoposide catechol in human plasma and CSF. The MNFs for plasma and CSF were tested by using one plasma sample and one CSF sample. The obtained MNFSTD-plasma and MNFSTD-CSF values were utilized to link the MEs of the plasma and CSF samples to the standard solution.
The MNF correction results are shown in Figure 3.2 Before correction, 500 ng mL-1 of etoposide standard solution showed the highest intensity (Figure 3.2 (a)), but the CSF etoposide showed a very small intensity due to its serious ME. Correction using the PCI-IS (Figure 3.2 (b)) partly corrected the MEs that caused the intensity differences, but they still were not fully compensated (Figure 3.2 (c)). Further correction using MNFSTD-CSF and MNFSTD-plasma gave very good ME correction, as indicated in Figure 3.2 (d).
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3.3.3. Validation of the PCI-IS method in combination with MNFs for quantifying
etoposide and etoposide catechol in plasma and CSF
The PCI-IS method in combination with MNFs was validated in terms of accuracy, linearity, precision and sensitivity. Etoposide and etoposide catechol-spiked plasma and CSF samples were used for method validation. Teniposide was used as the PCI-IS to correct the MEs. This is the first time that the PCI-IS method in combination with MNFs has been used to quantify target analytes in different biofluids, and we validated
the LC-ESI-MS method using drug-spiked plasma and CSF samples. For future studies that apply this approach, only standard solution need to be used for method validation.
3.3.3.1. Precision
The intra-day and inter-day precision were tested four times a day for 3 days at concentrations of 10, 150 and 500 ng mL-1 (Table 3.1). The intra-day precision (n = 12) and inter-day precision (n = 3) of etoposide in different spiked plasma and CSF samples had relative standard deviations (RSDs) less than 14%. The intra-day precision (n = 12)
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and inter-day precision (n = 3) of etoposide catechol in different spiked plasma and CSF samples had RSDs of less than 17%.
3.3.3.2. Quantification accuracy
Etoposide catechol and etoposide were spiked into 7 different blank plasma samples and 3 different blank CSF samples at concentrations of 10, 150, and 500 ng mL-1 to test for their recoveries (Figures 3.3 and 3.4). The results showed that more than 93% of the data have quantification errors of less than 20%, with 99% of the data having quantification errors of less than 30%.
3.3.3.3. Linearity, limits of quantification (LOQs) and limits of detection (LODs)
When using PCI-IS in combination with MNFs to quantify analytes in samples, the signals in the samples should be corrected by MNFs and PCI-IS and then quantified using a calibration curve generated by standard solutions. The linearity was tested using etoposide and etoposide catechol standard solutions at concentrations of 10, 50, 150, 250, and 500 ng mL-1. The calibration curves were y = 0.00003x2 + 0.0713x - 0.2805 (r2
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= 0.9999) for etoposide and y = 0.00001x2 + 0.0198x - 0.0164 (r2 = 0.9999) for etoposide catechol.
Because CSF has the most serious MEs, the LOD and LOQ scores were tested in the spiked CSF samples. The LOQs of etoposide and etoposide catechol were both 10 ng mL-1, and the LODs of etoposide and etoposide catechol were both 5 ng mL-1.
3.3.4. The advantages of using the PCI-IS method in combination with MNFs for
bioanalysis
Because isotope-labeled etoposide and etoposide catechol can only be obtained by customized synthesis, their price is relatively high. Currently, most bioanalysis methods for quantifying etoposide and etoposide catechol use structural analogs as an internal standard. Teniposide was selected as the internal standard in several studies due to its structural similarity with etoposide and etoposide catechol [22, 23]. Our previous study indicated that the retention time of teniposide was different from that of etoposide and etoposide catechol, which may have resulted in different extents of ME exposure.
PCI-IS can reveal a more correct chemical environment for the target analyte and improve the quantification accuracy.
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Moreover, the PCI-IS method in combination with MNFs is able to correct the MEs of various biofluids with distinct matrix compositions. Traditionally, to quantify a target analyte in different biofluids, different calibration curves had to be established and validated in each biofluid. As MNFs can effectively correct the difference in MEs between different biofluids and PCI-IS can additionally tailor the correction of the MEs for individual samples, the calibration curve generated by standard solutions is able to quantify the target analyte in various biofluids. The improvement in the quantification accuracy for multiple biofluids is demonstrated in Figures 3.3 and 3.4. Before correction, the raw data showed very poor accuracy for both plasma and CSF when using the calibration curve generated by the standard solutions. After correction by MNFSTD-plasma
and MNF STD-CSF, the quantification errors were partially corrected, and the quantification results were closer to the true values (Figure 3.3 (b1 and b2) and Figure 3.4 (b1 and b2)). By the additional correction of the PCI-IS signal for individual samples, over 93% of the data showed quantification errors of less than 20% (Figure 3.3 (a3 and b3) and Figure 3.4 (a3 and b3)). In summary, MNFs combined with the PCI-IS method largely simplify the entire method development and validation process, save much time and cost without sacrificing quantification accuracy, and provide a facile method for the quantification of target analytes in different biofluids.
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3.3.5. Application of MNFs in combination with the PCI-IS method to human samples
To demonstrate the feasibility of MNFs and the PCI-IS method for clinical measurement, plasma and CSF samples from one patient with a brain malignancy receiving etoposide treatment were analyzed during two treatment cycles. Samples were collected before dosing, 1 hour after dosing, 2 hours after dosing, and 6 hours after dosing. Figure 3.5 shows the pharmacokinetic profiles of etoposide and etoposide catechol in the CSF and plasma of this patient. The relatively high hydrophilicity of etoposide catechol hindered its penetration through the blood-brain barrier into the CSF, which resulted in its low concentration in the CSF samples. The CSF etoposide catechol concentration was lower than the LOQ and was therefore not quantified. Except for etoposide catechol in the CSF, we successfully applied the PCI-IS method in combination with MNFs to quantify etoposide and etoposide catechol in the plasma and CSF samples. Based on the CSF and plasma pharmacokinetic profiles during two treatment cycles, we observed a higher area under the curve of the CSF profile during the second treatment cycle. The patient was administered an anti-vascular endothelial growth factor (VEGF) drug, bevacizumab, before etoposide dosing in the second cycle.
The simultaneous analysis of the drug concentrations in the plasma and CSF samples
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facilitated the investigation of the effect of bevacizumab on the penetration of etoposide from the blood into the CSF to treat the brain tumor. Our proposed MNFs in combination with the PCI-IS method greatly simplified the bioanalysis for these complicated pharmacokinetic studies.
3.4. Conclusion
In this study, we developed an efficient strategy using a PCI-IS method in combination with MNFs to quantify target analytes in various biofluids. The implementation of MNFs in the PCI-IS method expanded the scope of this method for quantifying analytes in various types of biofluids existing in the real world. The MNFs were designed to associate the large differences in MEs between different biofluids, and the PCI-IS was used to additionally correct the MEs for individual samples. This approach allows the use of calibration curves generated by standard solutions to quantify the target analytes in various biofluids. We successfully applied MNFs to quantify etoposide and etoposide catechol in plasma and CSF samples. We anticipate that this new proposed strategy could improve the analytical quality and speed in the fields of bioanalytics, forensic toxicology, environmental pollutant studies and food safety.
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Table 3.1 The intra-day and inter-day precision.
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Figure 3.1 The electropherograms of plasma and CSF subjected to different PPT methods.
(a) A 1,000-fold dilution of the plasma sample; (b) plasma deproteinized by 4x volume of methanol; (c) plasma deproteinized by 2x volume of ACN; (d) CSF without any sample pretreatment; (e) CSF deproteinized by 4x volume of methanol; (f) CSF deproteinized by 2x volume of ACN.
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Figure 3.2 The MRM chromatograms of (a) 500 ng mL-1 of etoposide, (b) 100 ng mL-1 PCI-IS (teniposide), (c) etoposide corrected by the response ratio of etoposide to PCI-IS, and (d) etoposide after correction with etoposide to PCI-IS and MNFSTD-CSF or MNFSTD-plasma in standard, plasma, and CSF biofluids.
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Figure 3.3 The accuracy test results of etoposide catechol (a1, a2, and a3) and etoposide (b1, b2, and b3) before correction (a1 and b1), after correction by MNFs alone (a2 and b2), and after correction by both MNFs and the PCI-IS (teniposide) (a3 and b3) in plasma samples.
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Figure 3.4 The accuracy test results of etoposide catechol (a1, a2, and a3) and etoposide (b1, b2, and b3) before correction (a1 and b1), after correction by MNFs alone (a2 and b2), and after correction by both MNFs and the PCI-IS (teniposide) (a3 and b3) in CSF samples.
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Figure 3.5 The pharmacokinetic profiles of etoposide in CSF (a and b) and plasma (c and d) and etoposide catechol in plasma (e and f) obtained from a patient with a brain malignancy undergoing etoposide treatment.
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