The authors have declared that no competing interests exist.
Conceived and designed the experiments: JHY LHP. Performed the experiments: JHY. Analyzed the data: JHY LHP. Contributed reagents/materials/analysis tools: JHY LHP. Wrote the paper: JHY LHP.
Matrix effects (MEs) continue to be an obstacle in the development of the LC-MS/MS method, with phospholipids being the major cause of MEs. Changing the mobile phase has been a common strategy to reduce MEs; however, the underlying mechanism is unclear. "In-source multiple-reaction monitoring" (IS-MRM) for glycerophosphocholines (PCs) has been commonly applied in many bioanalytical methods. "Visualized MEs" is a suitable term to describe the application of IS-MRM to visualize the elution pattern of phospholipids. We selected a real case to discuss the relationship of MEs and phospholipids in different mobile phases by quantitative, qualitative, and visualized MEs in LC-MS/MS bioanalysis. The application of visualized MEs not only predicts the ion-suppression zone but also helps in selecting an appropriate (1) mobile phase, (2) column, (3) needle wash solvent for the residue of analyte and phospholipids, and (4) evaluates the clean-up efficiency of sample preparation. The TRAM-34 LC-MS/MS method, improved by using visualized MEs, was shown to be a precise and accurate analytical method. All data indicated that the use of visualized MEs indeed provided useful information about the LC-MS/MS method development and improvement. In this study, an integrative approach for the qualitative, quantitative, and visualized MEs was used to decipher the complexity of MEs.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a versatile technique in the field of life sciences. Its high sensitivity, specificity, accuracy, and robustness help in qualifying and quantifying the analytes in a biological matrix. However, matrix effects (MEs), resulting from coeluting matrix components, lead to unreliable results [
Phospholipids are the main components of the cell membranes and the major cause of MEs in bioanalytical methods [
(A) Structure of LPC. The ion fragments are
“Visualized MEs” is a suitable term to describe the application of IS-MRM to visualize the elution pattern of PC, LPC, and SM in the chromatogram. Many applications have been developed for the IS-MRM transition to monitor the elution of phospholipids. This study describes the integrative view of the qualitative, quantitative, and visualized MEs. A highly selective KCa3.1 channel blocker, 1-[(2-chlorophenyl) diphenylmethyl]-1
MEs present obstacles in the LC-MS/MS method development. Changing the mobile phase is a common strategy to attenuate MEs; however, the underlying mechanism remains unclear. In this study, three different mobile-phase B solutions (acetonitrile (ACN), methanol (MeOH), and MeOH/ACN 50/50) were used for their different elution properties for phospholipids and TRAM-34. First, we used the same SPE method and LC gradient elution profile with three different mobile-phase B solutions to investigate the relationship between TRAM-34, phospholipids, and MEs. Second, we used visualized MEs to improve the TRAM-34 method and the sample preparation method was changed from SPE to protein precipitation (PPT). Third, we shortened the gradient elution time from 7.5 min to 6 min with the help of visualized MEs. Finally, we evaluated the linearity, accuracy, precision, and qualitative MEs to verify the feasibility of the visualized MEs. Our integrated approach for qualitative, quantitative, and visualized MEs provided an appropriate model to unravel the relation between the MEs and the changing mobile phase.
TRAM-34 was purchased from Toronto Research Chemicals (Ontario, Canada). Bifonazole (
Six male nine-week-old Sprague–Dawley (SD) rats (BioLasco Taiwan Co., Ltd., Taipei, Taiwan) were used with approval from the Institutional Animal Care and Use Committee (IACUC) of National Defense Medical Center (NDMC) (IACUC-12-254). The housing, care, diet, and maintenance of the experimental animals were consistent with the recommendations of the National Research Council’s Guide for the Care and Use of Laboratory Animals, Animal Welfare Act, and NDMC Policy. The SD rats were 315 ± 10 g at nine weeks; they were anesthetized by Zoletil 50 (Carros Cedex, France) before performing the carotid artery catheterization. A blood sample was collected into a heparin-containing tube. The plasma was collected after the blood sample had been centrifuged at 12000 rpm for 10 min. These plasma samples were stored at −80°C.
The concentration of the TRAM-34 stock solution was 1 mg/mL in MeOH. The concentrations of the working standards were in the range 10 ng/mL–10 μg/mL; they were prepared by serial dilutions with MeOH. The concentration of the IS stock solution was 1 mg/mL; it was diluted to 0.2 μg/mL with 50% MeOH in water. Seven plasma calibration standards and three quality control (QC) samples were prepared by spiking the TRAM-34 working standard into the rat blank plasma. The final concentrations of the seven calibration samples were 1, 10, 100, 250, 500, 700, and 1000 ng/mL. Seven plasma calibration standards and three QC samples were prepared by spiking 10 μL of the TRAM-34 working standard into 90 μL of the rat blank plasma. Then, 100 μL of a 0.2 μg/mL IS solution was added into the TRAM-34-spiked rat plasma to yield the final 200 μL samples. The three QC samples were 3 ng/mL (low-concentration QC, LQC), 450 ng/mL (medium-concentration QC, MQC), and 800 ng/mL (high-concentration QC, HQC).
SPE was performed to extract TRAM-34 and bifonazole from the rat plasma [
The PPT method was performed to extract TRAM-34 and IS from the rat plasma. One hundred and fifty μL of the 0.167-μg/mL IS solution (in ACN) was added into the TRAM-34-spiked rat plasma to yield the final 200-μL samples. These calibration standards and QC samples were centrifuged at 12,000 rpm and 4°C for 10 min. Finally, 150 μL of the supernatant was transferred to the inserts of the autosampler vial for LC-MS/MS analysis.
The liquid chromatography (LC) system used was the Agilent 1200 series (Agilent Technologies, Santa Clara, USA). An XBridge BEH phenyl column (50 × 3 mm, 5 μm particle size, Waters, Milford, USA) and SecurityGuard phenyl guard cartridges (4 × 2.0 mm, Phenomenex, Torrance, USA) were used to perform the separation. The column oven temperature was 50°C.
The LC system was equipped with a triple quadrupole API 3200 LC-MS/MS system (AB Sciex, MA, USA). The Turbo V ion source of API 3200 was operated in positive-mode electrospray ionization (ESI), and the ion-spray voltage was 5 kV. The temperature of the ion source was 500°C. According to the 2002/657/EC guidelines, one precursor ion and two product ions could meet the criteria of compound identification (identification points, IP = 4) [
The reconstituted samples (processed by SPE) were transferred to the inserts of the autosampler vial for LC-MS/MS analysis. The LC conditions were as follows: mobile phase A, was 0.2% FA in water; mobile phase B, 0.2% FA in ACN/MeOH (1:1 v/v), 0.2% FA in MeOH, or 0.2% FA in ACN. The flow rate was 400 μL/min. The gradient elution was as follows: mobile phase A: 0–1 min, 70–30%; 1–1.5 min, 30–0%; 1.5–4.5 min, 0%; and 4.51–7.5 min, 70%. The run time of a single injection was 7.5 min. The autosampler temperature was 4°C. The sample injection volume was 5 μL.
The reconstituted samples (processed by PPT) were transferred to the inserts of the autosampler vial for LC-MS/MS analysis. Mobile phase A was 0.2% FA in water, and mobile phase B was 0.2% FA in ACN. The flow rate was 600 μL/min. The gradient elution was as follows: mobile phase A: 0–1 min, 70–30%; 1–1.5 min, 30–0%; 1.5–3.5 min, 0%; and 3.51–6 min, 70%. The run time of a single injection was 6 min. The autosampler temperature was 4°C. The sample injection volume was 10 μL.
The Q1 full scan (
The postcolumn infusion experiment was achieved by the constant infusion of 1000 ng/mL of TRAM-34 and 2000 ng/mL of IS. The Harvard infusion pump was used to infuse the TRAM-34 and IS solutions through a T-connector into the ion source at a speed of 10 μL/min. Under the constant infusion of the TRAM-34 solution, the chromatogram of TRAM-34 MRM transition (
The first set (Set 1) was defined as that containing TRAM-34 and IS obtained after spiking with the SPE-treated rat plasma. The second set (Set 2) was defined as that containing TRAM-34 and IS spiked with ACN/water (1:1 v/v) solution. There were six replicates of the QC samples in Set 1 and Set 2; the peak areas of TRAM-34 and IS were calculated for the quantitative MEs (Set 1/Set 2 × 100 (%)) [
Selectivity was evaluated by six rat blank plasma samples, collected from different sources. The lower limit of quantification (LLOQ) had an S/N ratio of >10. The linearity was assessed from the slope of seven nonzero points on the calibration curve. The weighting was 1/x. The linear range was 1–1000 ng/mL; the correlation coefficient was accepted when the value was >0.995.
The evaluation criteria of accuracy of the QC samples in the within-run and between-run tests was within ±15% range of the nominal concentration, while the criterion for the LLOQ was within ±20% of the nominal concentration. The precisions of the QC samples in the within-run and between-run tests were accepted when the coefficients of variation (CV) values were <15% (<20% for LLOQ) [
During the TRAM-34 LC-MS/MS method development with SPE-sample preparation, when mobile phase B was 0.2% FA in MeOH, the response between TRAM-34 and the internal standard (IS) showed a plateau in the curve (
Blue line represents the calibration curve; red line represents the plateau curve at high concentrations. calibration curve using ACN + 0.2% FA (r = 0.9996), (B). When comparing the linearity in Fig. 2A and 2B, it represents that there might be massive ion suppression in MeOH + 0.2% FA.
It was inferred that the plateau of the calibration curve did not result from saturated ionization. Initially, the signal was very high when we spiked the 50% MeOH solution with 1000 ng/mL TRAM-34. We then spiked the plasma with 1000 ng/mL TRAM-34, and the signal was significantly decreased. Finally, a plateau was observed in the curve when the number of injections was increased. The calibration curve without the influence of MEs in ACN + 0.2% FA showed good linearity (
Because Little
When mobile phase B is MeOH/ACN 50/50 with 0.2% FA (MP-B1), the elution peak of TRAM-34 was located in the trough of the elution pattern of the phospholipids (
(A) Mobile phase B is MeOH/ACN 50/50 + 0.2% FA. Relative elution time and elution pattern of TRAM-34 and phospholipids for both the
When using 100% MeOH with 0.2% FA (MP-B2), the elution patterns of TRAM-34 and the phospholipids overlapped (
Judging from these observations, it was necessary to examine the elution pattern of TRAM-34 and phospholipids with 0.2% FA in 100% ACN (MP-B3). Surprisingly, the elution of TRAM-34 was nearly free from these phospholipids (
The elution patterns of IS and the phospholipids in the three mobile-phase B solutions (MP-B1–B3) were monitored (
In summary, these observation in the three different mobile-phase B solutions demonstrated that the visualization of the phospholipid elution pattern might be of tremendous benefit to LC-MS/MS method development and improvement.
Postcolumn infusion is a common strategy to access the ion-suppression zone, which is affected by coeluting compounds, in an LC-MS/MS method. In order to verify whether the ion suppression results from the visualized patterns in the IS-MRM channels, postcolumn infusion experiments were performed. The continuous infusion of TRAM-34, the dip, compared to the injection of 50% ACN in water, represented the ion-suppression zone of TRAM-34 in the LC-MS/MS method. In the chromatogram of MP-B1 (
(A) Mobile phase B is MeOH/ACN 50/50 + 0.2% FA. There were four dips that represent ion suppression in the chromatogram. Asterisk: elution of TRAM-34 was located in the peak of ion-suppression zone. (B) Mobile phase B is MeOH + 0.2% FA. There were three dips that represent ion suppression in the chromatogram. Elution of TRAM-34 was located in the largest dip. (C) Mobile phase B is ACN + 0.2% FA. There were three dips that represent ion suppression in the chromatogram. Elution of TRAM-34 was not located in the dip.
There were three dips (arrows) in the chromatogram of MP-B2 (
Similarly, there are three dips (arrows) in the chromatogram of MP-B3 (
The postcolumn infusion data of IS are presented in
Interestingly, the same ion-suppression zone during the RT 0.5–1.5 min was observed in all the chromatograms of the qualitative MEs. In the early-elution zone, the ion suppression has been regarded as the product of salts, other nonretained components, and polar proteins and carbohydrates [
Given the elution patterns of the phospholipids in the IS-MRM chromatograms and the dips in the qualitative MEs, the visualized elution patterns of the phospholipids could predict the ion-suppression zone in the LC-MS/MS method, thereby providing useful information for the development of LC-MS/MS analytical methods.
As mentioned above, MEs resulted from the different mobile-phase B solutions influenced the linearity during the TRAM-34 analytical method development. A comparison of the chromatograms of the visualized MEs shows that qualitative MEs and quantitative MEs in three different mobile-phase B solutions could reveal the importance of the relationship of MEs with phospholipids in LC-MS/MS bioanalysis. To investigate the relation between the mobile-phase B solutions and the quantitative MEs, MP-B1, MP-B2, and MP-B3 were used to evaluate the quantitative MEs. The data of the quantitative MEs in the three different mobile-phase B solutions are listed in
ACN/MeOH 1:1 solution B (n = 6) | 100% MeOH solution B (n = 6) | 100% ACN solution B (n = 6) | ||||||
---|---|---|---|---|---|---|---|---|
Nominal concentration (ng/ml) | Calculated matrix effect (%) | Calculated matrix effect (%) | Calculated matrix effect (%) | |||||
TRAM-34 | Bifonazole | TRAM-34 | Bifonazole | TRAM-34 | Bifonazole | |||
3 | 46.1 ± 3.8 | 86.5 ± 3.9 | 33.3 ± 2.7 | 90.3 ± 2.1 | 113.9 ± 7.7 | 85.0 ± 3.9 | ||
450 | 74.6 ± 6.0 | 88.1 ± 5.5 | 64.4 ± 3.2 | 101.7 ± 7.6 | 110.1 ± 1.2 | 89.2 ± 2.6 | ||
800 | 73.8 ± 8.7 | 88.0 ± 5.2 | 62.6 ± 1.9 | 105.0 ± 3.1 | 106.7 ± 2.0 | 91.4 ± 2.3 |
The MEs of LQC, MQC, and HQC in MP-B3 were 113.9±7.7%, 110.1±1.2%, and 106.7±2.0%, respectively. It was interesting to compare the qualitative (
Visualized MEs is a suitable term to describe the application of IS-MRM to visualize the elution pattern of PCs, LPCs, and SMs. We also examined a real case to discuss the relationship of the MEs with phospholipids according to the quantitative, qualitative, and visualized MEs in the LC-MS/MS bioanalysis. The underlying mechanism of the change of solvent was revealed by performing an integrative examination of these three kinds of MEs. Different solvents possess various elution abilities for the analyte and phospholipids, which results in different MEs during LC-MS/MS analysis.
In LC-MS/MS, LPCs are more likely to produce MEs [
The
A precursor ion scan of
According to the above findings, the TRAM-34 LC-MS/MS method could be easily improved by employing the strategy of visualized MEs.
During LC-MS/MS method development, compound tuning, column selection, LC condition testing, method validation, and quantitative matrix effects assessment are necessary. To boost LC-MS/MS method development, use of visualized matrix effects is introduced. By monitoring the elution of the analyte and phospholipids, it is easier to evaluate whether the LC conditions are appropriate. Visualization of the phospholipid elution pattern is of tremendous benefit to LC-MS/MS method development and improvement.
By introducing visualized MEs (
With the correlation of visualized, qualitative, and quantitative MEs revealed in the present study, it is easier to improve LC-MS/MS methods. We modified the LC conditions and changed the sample preparation method using the strategy of visualized MEs. Although SPE was usually thought to result in fewer MEs, the present data showed that most phospholipids were not removed by SPE under these experimental conditions. Instead of removing phospholipids, the different elution sequence of the analyte and phospholipids provided new insight into how one can mitigate the MEs. Although the PPT method was more economic and time-saving, it would result in more MEs than SPE for sample preparation in general. After the visualized MEs were used to make sure that the elution sequence of TRAM-34 and phospholipids was not affected by PPT, the sample preparation was changed from SPE to PPT.
The “LC for SPE method” and “LC for PPT method” were used as two different analytical methods, and the results were compared. In the “LC for PPT method,” the flow rate was increased, and the elution sequence of the phospholipids and TRAM-34 in MP-B3 was unchanged (
To simultaneously wash the residue of TRAM-34 and phospholipids in LC system, various needle wash solvents were used: ACN, MeOH, IPA, MeOH/ACN 1:1, ACN/IPA 1:1, and MeOH/IPA 1:1 (Figure A and B in
In summary, the TRAM-34 LC-MS/MS analytical method was improved by using the strategy of visualized MEs. The improved “LC for PPT method” is summarized as follows: sample volume, 45 μL; sample preparation, PPT; needle wash solvent, MeOH/IPA 1:1; run time, 6 min; and flow rate, 600 μL/min. The LLOQ was still 1 ng/mL. Further method validation is needed to verify the feasibility of the strategy of visualized MEs in method development.
The data of the quantitative MEs in the “LC for PPT method” are listed in
Matrix effect data (n = 6) | ||
---|---|---|
Nominal concentration (ng/ml) | Calculated matrix effect (%) | |
TRAM-34 | Bifonazole | |
3 | 95.5 ± 1.4 | 92.0 ± 1.7 |
450 | 93.3 ± 0.6 | 96.8 ± 0.3 |
800 | 95.4 ± 0.6 | 95.8 ± 0.5 |
Set 1 = TRAM-34 and IS spiked in the extracted plasma.
Set 2 = TRAM-34 and IS spiked in the acetonitrile:water.
Matrix effect = Set 1/ Set 2 × 100
There are many commercial products to eliminate or decrease the MEs, but their high cost is a major issue for most labs. This study shows that changing the mobile phase could effectively avoid the phospholipid-induced MEs, representing a simple, fast, and economical strategy.
In summary, the quantitative MEs in this study were showed that the phospholipids in the visualized MEs were responsible for the massive ion suppression.
There were nearly no MEs when the elution of TRAM-34 was separate from the elution pattern of the phospholipids.
The selectivity was evaluated using six different rat plasma samples. Setting two MRM channels for the TRAM-34 provided good selectivity [
The accuracy and precision within-run and between-run in the “LC for PPT method” are listed in
Nominal concentration (ng/ml) | Within-run | Between-run | |||||
---|---|---|---|---|---|---|---|
Mean concentration (ng/ml) ± s.d. | Precision (CV, %) | Accuracy (%) | Mean concentrati on (ng/ml) ± s.d. | Precision (CV, %) | Accuracy (%) | ||
LLOQ | 1 | 1.0 ± 0.1 | 6.9 | 96.2 | 1.0 ± 0.1 | 8.3 | 99.4 |
LQC | 3 | 3.1 ± 0.1 | 2.0 | 104.7 | 3.0 ± 0.2 | 6.5 | 101.6 |
MQC | 450 | 467.2 ± 6.2 | 1.3 | 103.8 | 469.0 ± 8.4 | 1.8 | 104.2 |
HQC | 800 | 808.3 ± 10.0 | 1.2 | 101.0 | 839.5 ± 39.0 | 4.6 | 104.9 |
In this study, we showed the benefits of using visualized MEs in the development of LC-MS/MS methods. The application of visualized MEs could not only predict the ion-suppression zone but also helps in selecting an appropriate (1) mobile phase (2) column, (3) needle wash solvent for the residue of analyte and phospholipids, and (4) evaluate the clean-up efficiency of the sample preparation. The improved TRAM-34 LC-MS/MS method was precise and accurate. This study described the integrative view of the qualitative, quantitative, and visualized MEs for TRAM-34. This is an appropriate model to decipher the complexity of MEs by examining three kinds of MEs. Visualized MEs indeed provided useful information about the LC-MS/MS method development, validation, and improvement.
(A) The m/z 104.1 → 104.1 transition was tuned after this spectrum was acquired. It represents the existence and abundance of LPCs in the plasma. (B) The
(TIF)
Elution of IS in three different mobile phases. There was no significant interference by coeluted phospholipids with IS.
(TIF)
Qualitative matrix effects of IS in three different mobile phases. The number of dips was different under different relative conditions of IS. This indicated that the matrix effects were compound-dependent.
(TIF)
Comparison of IS-MRM transitions and qualitative matrix effects in the early elution. The coincidence of the peaks in IS-MRM transitions and dips in the qualitative matrix effects suggested that phospholipids might be responsible for ion suppression in the early elution.
(TIF)
There were 102 species of LPCs, PCs, and SMs identified by LipidView (AB Sciex).
(TIF)
Q1 full scan raw data were imported into LipidView software, and the lipid species were identified. Many peaks could not be identified by the LipidView software, which represents that phospholipids might not the only source to cause matrix effects in the LC-MS/MS bioanalysis. The LC conditions were as follows: mobile phase A, was 0.2% FA in water; mobile phase B, 0.2% FA in ACN/MeOH (1:1 v/v), 0.2% FA in MeOH, or 0.2% FA in ACN. The flow rate was 400 μL/min. The gradient elution was as follows: mobile phase A: 0–1 min, 70–30%; 1–1.5 min, 30–0%; 1.5–4.5 min, 0%; and 4.51–7.5 min, 70% (LC for SPE method).
(TIF)
(A) Five tentative phospholipids (
(TIF)
The RT of TRAM-34 in “LC for PPT method” was 3.28 ± 0.2 min. The elution time of TRAM-34 and PCs would slightly shift at the same time. The stability of RT was relatively stable. Elution sequence of phospholipids and TRAM-34 in 100% ACN was unchanged compared to
(TIF)
(A) IPA was the best wash solvent for TRAM-34. ACN was the worst. (B) MeOH was the best wash solvent for the phospholipids. ACN was the worst.
(TIF)
We acknowledge the Forensic Laboratory and the Department of Disaster Management, Taiwan Police College, for instrument support.