scholarly journals Necessity and Challenges of Sample Preconcentration in Analysis of Multiple MicroRNAs by Capillary Electrophoresis

2020 ◽  
Author(s):  
Liang Hu ◽  
Svetlana Krylova ◽  
Stanley K. Liu ◽  
George M. Yousef ◽  
Sergey Krylov
Keyword(s):  
Capillary Electrophoresis ◽  
Simultaneous Detection ◽  
Biological Matrices ◽  
Current Standard ◽  
Limit Of Quantification ◽  
Cell Lysates ◽  
Laser Induced Fluorescence Detection ◽  
Sample Preconcentration ◽  
Hybridization Probes ◽  
Limit Of Quantitation

Here we present the result of our work on achieving sub-pM limit of quantitation in direct quantitative analysis of multiple miRNAs by capillary electophoresis with laser-induced fluorescence detection. The PDF file contains the description of the results with figures, references, and supporting information. The <i>raw data.ZIP</i> file is the archive containing (i) Excel files of raw data used to build figures presented in the PDF file and (ii) the <i>red-me.PDF </i>file explaining how to read and use the Excel files. The <i>figure data.ZIP</i> file is the archive of all figure files in Origin and Adobe Illustrator formats. <div><b>Abstract:</b> Thousands of putative miRNA-based cancer biomarkers have been reported but none has been validated for approval by the Food and Drug Administration. One of the reasons for this alarming discrepancy is the lack of a method which is sufficiently robust for carrying out validation studies, which may require analysis of samples from hundreds of patients across multiple institutions and pooling the results together. Capillary electrophoresis (CE)-based hybridization assay proved to be more robust than reversed transcription polymerase chain reaction (the current standard) but its limit of quantification (LOQ) exceeds 10 pM while miRNA concentrations in cell lysates are below 1 pM. Thus, CE-based separation must be preceded by on-column sample preconcentration. Here we explain challenges of sample preconcentration for CE-based miRNA analyses and introduce a preconcentration method that can suit CE-based miRNA analysis utilizing peptide nucleic acid (PNA) hybridization probes. The method combines field-amplified sample stacking (FASS) with isotachophoresis (ITP). We proved that FASS-ITP could retain and concentrate both near-neutral PNA with highly-negatively charged PNA–miRNA hybrids. We demonstrated that preconcentration by FASS-ITP could be combined with the CE-based separation of the unreacted PNA probes from the PNA–miRNA hybrids and facilitate improvement in LOQ by a factor of 140, down to 0.1 pM. Finally, we applied FASS-ITP-CE for simultaneous detection of two miRNAs in crude cell lysates and proved that the method was robust when used in complex biological matrices. The 140-fold improvement in LOQ and the robustness to biological matrices will significantly expand the applicability of CE-based miRNA analysis, bringing it closer to becoming a practical tool for validation of miRNA biomarkers. <div><br></div></div>

scholarly journals Necessity and Challenges of Sample Preconcentration in Analysis of Multiple MicroRNAs by Capillary Electrophoresis

2020 ◽  
Author(s):  
Liang Hu ◽  
Svetlana Krylova ◽  
Stanley K. Liu ◽  
George M. Yousef ◽  
Sergey Krylov
Keyword(s):  
Capillary Electrophoresis ◽  
Simultaneous Detection ◽  
Biological Matrices ◽  
Current Standard ◽  
Limit Of Quantification ◽  
Cell Lysates ◽  
Laser Induced Fluorescence Detection ◽  
Sample Preconcentration ◽  
Hybridization Probes ◽  
Limit Of Quantitation

Here we present the result of our work on achieving sub-pM limit of quantitation in direct quantitative analysis of multiple miRNAs by capillary electophoresis with laser-induced fluorescence detection. The PDF file contains the description of the results with figures, references, and supporting information. The <i>raw data.ZIP</i> file is the archive containing (i) Excel files of raw data used to build figures presented in the PDF file and (ii) the <i>red-me.PDF </i>file explaining how to read and use the Excel files. The <i>figure data.ZIP</i> file is the archive of all figure files in Origin and Adobe Illustrator formats. <div><b>Abstract:</b> Thousands of putative miRNA-based cancer biomarkers have been reported but none has been validated for approval by the Food and Drug Administration. One of the reasons for this alarming discrepancy is the lack of a method which is sufficiently robust for carrying out validation studies, which may require analysis of samples from hundreds of patients across multiple institutions and pooling the results together. Capillary electrophoresis (CE)-based hybridization assay proved to be more robust than reversed transcription polymerase chain reaction (the current standard) but its limit of quantification (LOQ) exceeds 10 pM while miRNA concentrations in cell lysates are below 1 pM. Thus, CE-based separation must be preceded by on-column sample preconcentration. Here we explain challenges of sample preconcentration for CE-based miRNA analyses and introduce a preconcentration method that can suit CE-based miRNA analysis utilizing peptide nucleic acid (PNA) hybridization probes. The method combines field-amplified sample stacking (FASS) with isotachophoresis (ITP). We proved that FASS-ITP could retain and concentrate both near-neutral PNA with highly-negatively charged PNA–miRNA hybrids. We demonstrated that preconcentration by FASS-ITP could be combined with the CE-based separation of the unreacted PNA probes from the PNA–miRNA hybrids and facilitate improvement in LOQ by a factor of 140, down to 0.1 pM. Finally, we applied FASS-ITP-CE for simultaneous detection of two miRNAs in crude cell lysates and proved that the method was robust when used in complex biological matrices. The 140-fold improvement in LOQ and the robustness to biological matrices will significantly expand the applicability of CE-based miRNA analysis, bringing it closer to becoming a practical tool for validation of miRNA biomarkers. <div><br></div></div>

scholarly journals Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry

2020 ◽  
Vol 58 (5) ◽  
pp. 836-846 ◽  
Author(s):  
Christine L. Skaggs ◽  
Greta J. Ren ◽  
El Taher M. Elgierari ◽  
Lillian R. Sturmer ◽  
Run Z. Shi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Therapeutic Drug ◽  
Reference Laboratory ◽  
Triple Quadrupole Mass Spectrometer ◽  
Limit Of Quantification ◽  
Paper Spray ◽  
Calibration Curves ◽  
Drug Concentrations ◽  
Limit Of Quantitation ◽  
Anti Fungal

AbstractBackgroundInvasive fungal disease is a life-threatening condition that can be challenging to treat due to pathogen resistance, drug toxicity, and therapeutic failure secondary to suboptimal drug concentrations. Frequent therapeutic drug monitoring (TDM) is required for some anti-fungal agents to overcome these issues. Unfortunately, TDM at the institutional level is difficult, and samples are often sent to a commercial reference laboratory for analysis. To address this gap, the first paper spray-mass spectrometry assay for the simultaneous quantitation of five triazoles was developed.MethodsCalibration curves for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were created utilizing plasma-based calibrants and four stable isotopic internal standards. No sample preparation was needed. Plasma samples were spotted on a paper substrate in pre-manufactured plastic cartridges, and the dried plasma spots were analyzed directly utilizing paper spray-mass spectrometry (paper spray MS/MS). All experiments were performed on a Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer.ResultsThe calibration curves for the five anti-fungal agents showed good linearity (R2 = 0.98–1.00). The measured assay ranges (lower limit of quantification [LLOQ]–upper limit of quantitation [ULOQ]) for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were 0.5–50 μg/mL, 0.1–10 μg/mL, 0.1–10 μg/mL, 0.1–10 μg/mL, and 0.1–10 μg/mL, respectively. The inter- and intra-day accuracy and precision were less than 25% over the respective ranges.ConclusionsWe developed the first rapid paper spray-MS/MS assay for simultaneous quantitation of five triazole anti-fungal agents in plasma. The method may be a powerful tool for near-point-of-care TDM aimed at improving patient care by reducing the turnaround time and for use in clinical research.

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