임상연구에 사용되는 질량분석 장비의 이해와 응용 내분비 연구팀 곽 호 석

질량 분석기 (Mass Spectrometry) Bioanalytical Method Validation –Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research May 2001 LC/MS 응용

Mass spectrometer Spectrophotometer A spectrometer is an instrument used to measure properties of light over a specific portion of the electromagnetic spectrum, typically used in spectroscopic analysis to identify materials. lightelectromagnetic spectrumspectroscopic analysis Mass Spectrometer

Inlet Ion Source Mass Analyzer Detector Data System High Vacuum System Mass Spectrometer Block Diagram

Inlet Ion Source Mass Analyzer Detector Data System High Vacuum System HPLC GC Sample plate (Dried sample) Sample Introduction

Inlet Ion Source Mass Analyzer Detector Data System High Vacuum System MALDI ESI EI CI ICP Ion Source

Inlet Ion source Mass Analyzer Detector Data System High Vacuum System Time of flight (TOF) Quadrupole Ion Trap FTMS Mass Analyzer

Inlet Gas Chromatography (GC) High Performance Liquid Chromatography (HPLC)

Ionization Liquid Solid Gas

Hard Ionization

Inductively coupled Plasma (ICP) Sample flow Magnetic field Induction coils Plasma Emission region Quartz tubes

Electron Ionization (EI)

Chemical Ionization (CI)

Soft Ionization

Electrospray Ionization (ESI) Atmospheric Pressure Ionization (API)

Atmospheric Pressure Chemical Ionization (APCI)

Matrix Assisted Laser Desorption Ionization (MALDI)

Examples (Soft Ionization)

The typical sensitivity and mass ranges allowed by different ionization techniques

Mass Analyzer

Quadrupole Mass Analyzers

Ion Trap Mass Analyzer Top View Cut away side view

Time of Flight (TOF) Mass Analyzer Reflector Detector Linear Detector Lens Target Extraction Plate Flight Tube Entrance Reflectron 337 nm Nitrogen laser

How do mass spectrometers get their names? Types of ion sources: Electrospray (ESI) Matrix Assisted Laser Desorption Ionization (MALDI) Types of mass analyzers: Quadrupole (Quad, Q) Ion Trap Time-of-Flight (TOF) -Either source type can work with either analyzer type: “MALDI- TOF,” “ESI-Quad.” -Analyzers can be combined to create “hybrid” instruments. ESI-QQQ, MALDI QQ TOF, Q Trap

QSTAR TM ESI QQ TOF or MALDI QQ TOF Q1 Ion Mirror (reflector) Effective Flight Path = 2.5 m Q2 Q0 Sample

What is MS/MS? MS/MS peptide selected for MS /MS The masses of all the pieces give an MS/M S spectrum Peptide mixture Have only masses t o start

Pharmaceutical analysis Bioavailability studies Drug metabolism studies, pharmacokinetics Characterization of potential drugs Drug degradation product analysis Screening of drug candidates Identifying drug targets Biomolecule characterization Proteins and peptides Oligonucleotides Environmental analysis Pesticides on foods Soil and groundwater contamination Forensic analysis/clinical Applications of Mass Spectrometry

The polarity and mass ranges allowed by different ionization techniques

BIOANALYTICAL METHOD VALIDATION

Matrix Biological matrix: A discrete material of biological origin that can be sampled and processed in a reproducible manner. –Examples are blood, serum, plasma, urine, feces, saliva, sputum, and various discrete tissues. Matrix effect: The direct or indirect alteration or interference in response due to the presence of unintended analytes (for analysis) or other interfering substances in the sample.

Recommended Validation Characteristics of the Various Types of Tests

Selectivity The ability of the bioanalytical method to measure and differentiate the analytes in the presence of components Selectivity should be ensured at the lower limit of quantification (LLOQ).

Accuracy The degree of closeness of the determined value to the nominal or known true value under prescribed condition. Accuracy should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentration is recommended. The mean value should be within 15% of the actual value except at LLOQ, where it should not deviate by more than 20%.

Precision The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed condition. Precision should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentration is recommended. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV.

Recovery The extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the sample extraction and processing steps of the method. Recovery of the analyte need not be 100%, but the extent of recovery of an analyte and of the internal standard should be consistent, precise, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high) with unextracted standards that represent 100% recovery.

Calibration (Standard) curve A calibration curve should consist of) a zero(blank) sample (matrix sample processed with internal standard), and six to eight non-zero samples covering the expected range, including LLOQ) The following conditions should be met in developing a calibration curve: –20% deviation of the LLOQ from nominal concentration –15% deviation of standards other than LLOQ from nominal concentration At least four out of six non-zero standards should meet the above criteria, including the LLOQ and the calibration standard at the height concentration.

Quantification range (limit of linearity)

LOD and LOQ

Sample A generic term encompassing controls, blank, unknowns, and processed samples, as described below: –Blank: a sample of a biological matrix to which no analytes have been added that is used to assess the specificity of the bioanalytical method. –Quality control sample (QC): a spiked sample used to monitor the performance of a bioanalytical method and to assess the integrity and validity of the result of the unknown samples analyzed in an individual batch. –Unknown: a biological sample that is the subject of the analysis.

Stability The chemical stability of an analyte in a given matrix under specific conditions for given time intervals. –Freeze and thaw stability At least three aliquots at each of the low and high concentrations should be stored at the intended storage temperature for 24hours and thawed unassisted at room temperature. –Short-term temperature stability Three aliquots of each of the low and high concentrations should be thawed at room temperature and kept at this temperature from 4 to 24 hours and analyzed. –Long-term stability The storage time in a long-term stability evaluation should exceed the time between the date of first sample collection and the date of last sample analysis. –Stock solution stability The stability of stock solutions of drug and the internal standard should be evaluated at room temperature for at least 6 hours. –Post-preparative stability The stability of the drug and the internal standard should be assessed over the anticipated run time for the batch size in validation samples by determining concentrations on the basis of original calibration standards.

LC/MS 응용

FAEE (Fatty acid ethyl ester)

FAEE 16:0 MH+ FA H+

FAEE 16:1 MH+ [FA-OH]+ [FA-OOH]+

Multiple Reaction Monitoring (MRM) Fixed Mass scan (285.5) (283.4) Collision cell Fixed Mass scan (257.7) (219.4) FAEE 16:0 FAEE 16:1

FAEE Standard E12:0 E16:0 E18:1 E18:2 E14:0 E16:1 E20:4 E18:3 E18:0 E17:0

Lysophosphatidic acid (LPA)

Product ion scan of LPA [M-H]-

Precursor Ion Scan Mass scan ( ) Collision cell Fixed Mass scan (153.1)

Precursor ion scan spectrum 개발된 추출 용액 사용 스펙트럼 기존의 추출 용액 사 용 스펙트럼 LPA C16:0 LPA C18:2 LPA C18:1 LPA C20:4 LPA C22:6

The End