The New 6495 Triple Quadrupole LC/MS
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1 The New 6495 Triple Quadrupole LC/MS Experience A New Level of Confidence Martin Haex Product Specialist MS and Automation 1
2 Overview of Topics The New 6495 QQQ LC/MS Technology Innovations Quantitation Performance - New performance standard IDL Key Applications New Software Capabilities Summary The New 6560 IM-QTOF LCMS Technology innovations Applications. Summary 2
3 Proven ifunnel Technology Agilent Jet Stream Hexabore Capillary Dual Ion Funnel Thermal gradient focusing Efficient desolvation Creates an ion rich zone Six capillary inlets Samples x10 times more ion rich gas Removes the gas but captures the ions Removes neutral noise 3
4 6495 QQQ Technologies Continued Development Proven ifunnel Technology Agilent Jet Stream Hexabore Capillary Dual Ion Funnel Increased ion generation Enhanced ion sampling New Enhanced Q1 Ion Optics 1 Improved ion transmission New Curved and Tapered Hexapole Collision Cell 2 Effective ion collection and transmission New Detector with High Energy Conversion Dynode 3 Improved NEG ion detection with low noise 4
5 6495 QQQ - Premium Performance Improved sensitivity and lower LLOQs Average 3x in S/N specifications and applications Improved precision and excellent accuracy at the lowest levels 3x in IDL specifications Proven 6 orders of linear dynamic range Proven robustness in complex matrix Food matrix and biological matrix (plasma) Improved mass range, fast scan speed and MRM acquisition rate 5
6 Improved Sensitivity Peak Area Response and S/N Reserpine (+) and Chloramphenicol (-), 1 pg on-column 1 pg reserpine (+) (n=10) 1 pg chloramphenicol (-) (n=10) x x higher area response 2.5x higher S/N 6495 Avg. Area = 14,519 Area RSD [%] = 1.5 Avg. S/N = 393,000: Avg. Area = 5,581 Area %RSD = 1.7 Avg. S/N = 157,000: Counts vs. Acquisition Time (min) x x higher area response 2.5x higher S/N 6495 Avg. Area = 8,808 Area RSD [%] = 1.1 Avg. S/N = 315,000: Avg. Area = 3,318 Area %RSD = 2.5 Avg. S/N = 125,000: Counts vs. Acquisition Time (min) The 6495 QQQ LC/MS system shows improved peak area response and S/N in both positive and negative ion modes compared to the previous designs 6
7 Many Ways to Manipulate S/N Increase signal Increase the gain Narrow chromatographic peak width Increase scan averaging Lower noise Select noise region Narrow the width of noise region Adjust baseline Apply peak smoothing & noise filtering Vary noise calculation algorithms: Peak-to-Peak, RMS, and... a. Peak-to-peak noise b. RMS noise c. Smoothing d. Baseline noise filtering Variation in S/N measurements makes direct assessment difficult 7
8 Instrument Detection Limit (IDL) is Defined by Statistics IDL LCMS = t x SD = t x (%RSD / 100) x amount measured Based on a well-established statistical formula, follows regulatory guidelines IDL The minimum amount of analyte that is detectable and distinguishable from background noise with a confidence level %RSD vs. Injected Amount t Student t value, for 99% confidence level n 1 degree of freedom %RSD Amount measured Relative standard deviation / precision of peak area at the amount measured From n replicate injections Limited to 2 5 x times higher than the Detection Limit (DL) Theoretical fitting of %RSD is based on ion statistics %RSD increases at lower injected amount 8
9 Why Change from S/N to IDL for QQQ LC/MS? Goal of Quantitative Analysis Reporting Detectable AMOUNT Sensitivity Performance of QQQ LC/MS Lowest detectable amount Detection Limit (DL) Lowest quantifiable amount Limit of Quantitation (LLOQ) Criteria of precision and accuracy are met at LLOQ Signal to Noise (S/N) NOT AMOUNT Variation associated with S/N (noise) measurement Fails to estimate the true detection limit and LLOQ, when measured at a high level of S/N > 50:1 Not a good metric of sensitivity performance Instrument Detection Limit (IDL) AMOUNT A rigorous metric based on statistical analysis of precision (%RSD) Measured at an analytical low level close to the detection limit Accurate assessment of the true detection limit and LLOQ A practical means of evaluating sensitivity performance 9
10 6495 QQQ Performance Specifications Bid Specifications Sensitivity, S/N Positive, 1 pg Reserpine Sensitivity, S/N Negative, 1 pg chloramphenicol Sensitivity, IDL Positive, Reserpine Sensitivity, IDL Negative, chloramphenicol Agilent 6490 Agilent ,000:1 150,000:1 50,000:1 150,000:1 2.5 fg 0.75 fg 2.5 fg 0.75 fg Linear dynamic range Mass range 5 1,400 Da 5 2,250 Da Maximum scan speed 12,500 Da/sec 15,000 Da/sec Maximum MRM rate 250 MRMs/sec 500 MRMs/sec Minimum MRM dwell time 1 ms 1 ms Polarity switching 30 ms 30 ms Installation Specifications Sensitivity, S/N Positive, 1 pg Reserpine Sensitivity, S/N Negative, 1 pg chloramphenicol 25,000:1 75,000:1 25,000:1 75,000:1 Improved sensitivity specs (3x) - S/N - IDL Improved mass range Improved full scan and MRM speed 10
11 Improved Sensitivity and Precision Peak Area %RSD Reserpine (+) and Chloramphenicol (-) at low levels, 5 fg on-column 5 fg reserpine (+) (n=10) 5 fg chloramphenicol (-) (n=10) x x higher area response Lower area %RSD 6495 Avg. Area = 157 Area %RSD = Avg. Area = 47 Area %RSD = Counts vs. Acquisition Time (min) 3 x higher area response Lower area %RSD x Avg. Area = 121 Area %RSD = Avg. Area = 40 Area %RSD = Counts vs. Acquisition Time (min) The improved sensitivity of the 6495 LC/MS system results in enhanced peak area response and improved area precision especially at the low levels This ultimately leads to lower detection limits (IDLs) and LLOQs compared to previous designs 11
12 Sensitivity and Precision Instrument Detection Limit Average of 13x 6495 units 6495 IDL Amount measured Replicates Area %RSD t (99%) IDL Reserpine (+) 1 fg 5 fg n = % 9.2% fg 1.3 fg Chloramphenicol (-) 1 fg 5 fg n = % 9.2% fg 1.3 fg 1 fg of Reserpine used to measure IDL (+) 1 fg of Chloramphenicol used to measure IDL (-) IDL = t x (%RSD / 100) x Amount = x (10.9 / 100) x 1 fg = 0.31 fg IDL = t x (%RSD / 100) x Amount = x (10.0 / 100) x 1 fg = 0.28 fg Area %RSD = 10.9 n = 10 injections Area %RSD = 10.0 n = 10 injections 1.4 Better precision and IDL (> 3x) on the 6495 vs. the 6490 Better precision and IDL (> 3x) on the 6495 vs. the
13 6495 QQQ Key Applications Food Safety - Pesticides Analysis of >250 trace level pesticides in food matrices (e.g. black tea) with extensive sample dilutions Food Safety - Estrogens Determination of ultra-trace level estrogens in milk product Environmental - Water Analysis Quantitation of ultra-trace level hormones (EDCs) in drinking water using direct injection Peptide Quantitation Quantitation of synthetic peptide at sub-attomole level using nanoflow and standard flow chromatography Peptide Quantitation Quantitative analysis of microcystins (cyclic nonribosomal peptides) in liver extract samples using stand flow chromatography Application tests are designed based on the requirements of the key high-end QQQ markets 13
14 FOOD SAFETY DETERMINATION OF ESTROGENS IN MILK Sensitivity, Precision, Accuracy, Linear Dynamic Range and Matrix Recovery 14
15 Sensitive Quantitation of Estrogens in Cow Milk ID Compounds Eight estrogens spiked in cow milk at 0.1 µg/kg, corresponding to 0.1 ng/ml 1 Estriol (-) 2 17-α-Estradiol (-) ng/ml estrogens in cow milk --- Milk blank Injection volume = 5 µl 7, β-Estradiol (-) 6 4 Ethynyl estradiol (-) 5 Estrone (-) 6 Diethylstilbestrol (-) 7 Hexestrol (-) Dienestrol (-) Estrogen levels in milk products are regulated by government agencies down to 1 µg/kg Highly sensitive and precise quantitation of estrogens was achieved using the new 6495 with a simple QuEChERS procedure without derivatization or enrichment. LLOQs for all estrogens (<< 0.1 µg/kg) are far below the regulatory requirement (1 µg/kg) 15
16 Estriol: Superb Precision and IDL at Low-fg Level Estriol Amount measured Replicates %RSD t (99%) Estriol IDL 5 pg/ml, 25 fg (LLOQ) n = 7 injections pg/ml, 8.9 fg IDL = t x (%RSD/100) x Amount = x (10.4/100) x 25 fg = 8.9 fg Injection volume = 5 µl 5 pg/ml 5 pg/ml 5 pg/ml 5 pg/ml 5 pg/ml 5 pg/ml 5 pg/ml Inj # Peak Area S/N (Peak to peak) %RSD 11.3 Ave Excellent peak area precision (%RSD) are observed at the lowest levels (LLOQs) 16
17 Confident Estrogen Quantitation in Milk, µg/kg Estriol in milk, µg/kg ng/ml R 2 = Estradiol in milk, µg/kg ng/ml R 2 = Levels (µg/kg) %RSD (n = 7) %Accuracy Levels (µg/kg) %RSD (n = 7) %Accuracy Excellent linearity (R ), accuracy and precision are achieved at all levels 17
18 Summary of Estrogen Quantitation in Milk Excellent linearity, precision, accuracy and matrix recovery Compounds R 2 Precision %RSD, n = 7 %Accuracy %Recovery 0.2 µg/kg 1 µg/kg 5 µg/kg Estriol α-Estradiol β-Estradiol Ethynyl estradiol Estrone Diethylstilbestrol Hexestrol Dienestrol
19 ANALYSIS OF HORMONES Sensitivity, Precision, Accuracy, Linear Dynamic Range, and Direct Injection 19
20 Analysis of Hormones Hormones in drinking water, 5 ng/l (testosterone) to 17.5 ng/l (EE) Testosterone 17- -Ethynyl estradiol (-) Estriol (-) Androstenedione 17-α-Ethynylestradiol Equilin Estrone 17- -estradiol (-) Equilin (-) Estriol 17-β-Estradiol Androstenedione (+) Testosterone (+) Estrone (-) Endocrine Disrupting Chemical (EDCs) levels in municipal water supplies are regulated by government agencies down to ng/l (ppt) level The highly sensitive 6495 allows the quantitation of EDCs at sub ng/l using direct injection No need for time consuming offline SPE or enrichment 20
21 Highest Sensitivity and Excellent Precision LLOQs and IDLs at sub-ng/l Level Compounds LLOQ (ng/l) %RSD n = 8 IDL (ng/l) % Accuracy Estriol β-Estradiol < Testosterone α-Ethynylestradiol Equilin < Estrone < Androstenedione < Estriol 2 ng/l Testosterone 0.1 ng/l 17β-Estradiol 0.5 ng/l 17 - Ethynylestradiol 1.75 ng/l Androstenedione 0.2 ng/l Equilin 0.2 ng/l Estrone 0.2 ng/l 21
22 17 -Estradiol: Superb Precision and Ultra-Low IDL Estriol Amount measured Replicates %RSD t (99%) IDL 0.5 ng/l (LLOQ) n = 8 injections ng/l MDL = t x (%RSD/100) x Amount = x (13.5/100) x 0.5 ng/l = ng/l 0.5 ng/l 0.5 ng/l 0.5 ng/l 0.5 ng/l 0.5 ng/l 0.5 ng/l Inj # Peak Area S/N (Peak to peak) 0.5 ng/l 0.5 ng/l %RSD Excellent peak area precision (%RSD) are observed at the lowest levels (LLOQs) 22
23 Confident Quantitation of EDCs in Drinking Water 17 -estradiol (-) ng/l R 2 = Testosterone (+) ng/l R 2 = Levels (ng/l) %RSD (n = 8) %Accuracy Levels (ng/l) %RSD (n = 8) %Accuracy Excellent linearity (R ), assay accuracy and precision are achieved at all levels 23
24 PEPTIDE QUANTITATION Sensitivity, Precision, Accuracy, Linear Dynamic Range and Robustness in Matrix 24
25 Outstanding Sensitivity with Standard Flow Chromatography Blank 3 amol Injection volume = 1 µl LOD LVNEVTEFAK 5 amol 5 pmol 6 Orders R 2 = Zoom-in 5 30 amol 5 amol Area %RSD = 14.0, n = 10 %Accuracy = LLOQ 7.5 amol Low attomole sensitivity for synthetic peptide spiked in tryptic digest using the 6495 with standard flow chromatography Six orders of linear dynamic range Excellent precision and accuracy are observed at all levels including the LLOQ level Levels %RSD (n = 10) % Accuracy 5 amol amol amol amol fmol fmol fmol fmol pmol pmol RT %RSD (n = 100)
26 Ultimate Sensitivity with Nanoflow LC Blank 0.25 amol LVNEVTEFAK 0.5 amol 0.1 pmol > 5 Orders R 2 = amol Area %RSD = 4.52, n = 5 %Accuracy = Zoom-in amol 1.0 amol Sub-attomole sensitivity for synthetic peptide spiked in tryptic digest using the 6495 with nanoflow chromatography Wide (over 5 orders) linear dynamic range Excellent precision and accuracy are observed at all levels including the LLOQ level Levels (amol) %RSD n = 5 Accuracy [%] , , RT %RSD n =
27 MassHunter Acquisition B.07 Support for 6495 QQQ LC/MS New diagnostics tool Nebulizer, PCA main board, quad driver, detector Acquisition Quant Faster and accurate autotune Improved workflows Optimizer to better support tmrm dmrm, tmrm, ifunnel express, Study Manager, Skyline automation, application kits and solutions 27
28 New Diagnostics Tool Lab Advisor Improve Serviceability and Reduce Warranty Costs Currently for MEs, FSEs and AEs + Mind map tool to guide users to diagnostic tests Lab Advisor tool to run tests and display results and reports Ion Source Quads / Quad Drivers Main PCA board Detectors Nebulizer Test Quad transmission and Quad Stability RFPA Quad frequency ramp Ramp gain and Ramp offset Faults Power supplies Ramp EMV power Ramp HED power Detector noise 28
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