Bioanalytical Method Development Strategy for Therapeutic

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Sam p l e P re pa rat ion, Oasis SPE sorbents are unique in their combination of purity reproducibility stability and retention. characteristics T he use of the extraction selectivity of two of the Oasis mixed mode ion exchange. sorbents in combination with a simple extraction protocol is a key contributor to the success rate of. this generic method development approach, T he innovative Oasis Elution plate format allows for up to a 15 sample concentration increasing. the possibility of reaching the required sensitivity levels for bioanalytical assays The low 25 L elution. volume eliminates the need for evaporation and reconstitution significantly reducing the potential. analyte loss due to absorption to the walls of the collection plate and or chemical instability. BEH Technology is Waters patented second generation hybrid particle that combines the efficiency. and structural integrity of silica particles with the pH stability of polymeric resins T he 300 C18. version of this particle delivers excellent peak shape for a wide range of peptide molecular weights. and is ideally suited for this generic approach, ACQUITY UPLC Technology is now firmly established as the LC platform of choice for bioanalytical. assays Holistically designed to maximize the potential of the BEH sub 2 m particle technology the. resultant chromatographic resolution sensitivity and sample throughput are fundamental contributing. factors to the accuracy and reproducibility of pharmacodynamic data. Mass Sp ec t romet ry, Xevo TQ MS is a highly advanced tandem quadrupole mass spectrometer Its unique collision cell. technology and extended capabilities allow simultaneous and multifunctional data acquisition within. a timescale compatible with UPLC Technology T he 2000 amu range of the first resolving quadrupole. represents a critical element for the generic applicability of this approach. T he combination of these unique novel technologies facilitates the generation of. method development solutions for extremely complex analytical challenges. Waters Xevo System with Peptide Separation Technology Consumables. Creating a Universal Approach to a Complex Problem. T he desire to commercialize peptides as therapeutic drug candidates creates a new series of challenges for the bioanalytical chemist in developing. simple robust and sensitive quantitative assays in support of clinical trial activities. Desmo p ressin Ac etat e, A successful generic bioanalytical assay needs to be.
Applicable to a diverse range of therapeutic peptides H HN. Selective and free from significant matrix interferences H. Linear over a wide dynamic range H O O,Capable of supporting high throughput assays. SCH2CH2C Tyr Phe Gln Asn Cys Pro D Arg Gly NH2 CH3COOH 3H2 0. MRM of 1 Channel ES,Blank Plasma,MRM of 1 Channel ES. 20 pg mL 21 4 fmol mL,Desmopressin in human plasma. 104 SPE Recovery,Matrix Effects 5, 0 2 0 4 0 6 0 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 min. A sensitive LC MS MS method for the extraction and analysis of Desmopressin from human plasma was developed utilizing a simple universal method development strategy T his method. could easily support an LLOQ of 20 pg mL and was demonstrated to have a linear response over 4 orders of magnitude. The Detection Challenge, Bioanalytical laboratories are extremely familiar with the use of triple quadrupole mass spectrometers for the routine analysis of biological.
samples However as the molecular weight of the analytes increases there are several characteristics of the particular mass spectrometer. chosen for this application that are critical to success. Ident if ying t he P recu rsor, T herapeutic peptides when analyzed by mass spectrometry can exist in multiple charge states such as 2 3 and 4 As the triple quadrupole. mass spectrometer detects analytes by their mass to charge ratio m z this does allow for the detection of peptides with a molecular weight. greater than the mass range of the spectrometer A successful highly sensitive generic MS method for the analysis of peptides relies upon the. detection of the major precursor and may be limited by the operating range of the first resolving quadrupole. 1498 32 1498 32Enfuvirtide,100 100 100 100,Scan ES Scan ES. Scan ES 1498 64 mw 4492,1498 01 1498 64,1494 1495 1496 1497 1498 1499 1500 1501 1502 1503. Enfuvirtide mw 4492 has possible precursor m z values of 1498 for 3 charge state. 400 600 800 1000 1200 1400 1600 1800 and 1124 0 for 4 charge state Under the analytical conditions used the 3 was the most. intense charge state 1495,present 1496,very little of the 4 1497 1498 1499 1500. was observed 1501 1502,400 600 800 1000 1200 1400 1600 1800.
Opt imizing for F ragmentat ion,Bivalirudin, T he ultimate sensitivity of the triple quadrupole MS detection method 100. depends upon the ability to optimize for and detect the most intense Doubly charged precursor. fragment s T he operating range of the second quadrupole should be Singly charged fragments. able to accommodate large fragment ions as seen in the example 1530. here and fragment m z values which are higher than the precursor. MRM Transitions identified, Bivalirudin mw 2180 MS scan shows 2 precursor present at m z 1091 1091 1530. After performing MSMS of m z 1091 from 100 to 1900 major fragments are observed. at m z 650 and m z 1530 Precursor appears at lower m z even though mw is higher 0 m z. 400 600 800 1000 1200 1400 1600, Note If need be area counts from several MRM transitions can be summed to. increase sensitivity,C h romatogra phic P erformanc e. A key parameter in the success of a generic method development approach is the correct choice of LC instrumentation and associated column chemistry. T he combination of ACQUITY UPLC and the 1 7 m BEH 300 C18 PST column results in excellent peak shape and chromatographic resolution for a. wide range of peptide molecular weights T he generic 3 5 minute gradient chromatographic method generates peak widths of less than 3 seconds. at base even for peptides with mw 4000 MS data acquisition of 12 points across each peak ensures accurate and reproducible quantitation. Analyte MW Peak Width MS Data Points,seconds Across Peak.
4 MRM of 5 Channels ES 1 Vasopressin 1084 1 8 15,2 Angiotensin II 1046 2 2 15. 3 Desmopressin 1069 2 2 18,4 Bivalirudin 2180 2 4 18. 5 Enfuvirtide 4492 2 1 16, ACQUITY UPLC BEH300 C18 2 1 X 50 mm 1 7 m Peptide Separation. Technology Column Mobile phase A 0 1 formic acid Mobile phase B. acetonitrile Flow rate 0 4 mL min 5 B to 75 B over 2 minutes. Total run time 3 5 minutes, 0 2 0 4 0 6 0 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 min. Sample Preparation Challenge, T he selective extraction of peptides from other endogenous biological components is probably.
one of the most challenging sample preparation tasks Approaches such as ELISA Ligand. Binding Assays are effective but are time consuming to develop and are not ideally suited. to routine bioanalytical DMPK studies T he technique of protein precipitation or conventional. reversed phase SPE although very common for this type of assay suffers from significant. ion suppression due to matrix effects thus impacting the achievable limits of detection. Sel ec t iv e Sam p l e E x t rac t ion, The Oasis family of SPE products are based upon a polymeric water wettable reversed phase sorbent with a usable pH range of 0 14 After extensive. analytical method development studies with a diverse range of therapeutic peptides Oasis WCX weak cation exchanger and Oasis MAX mixed mode. anion exchanger showed the greatest utility for the selective extraction from human plasma in terms of recovery and reduced matrix effects. A simple generic extraction protocol has been developed for these sorbents which when applied to the target list of peptides achieved 80 recovery. for 9 of the 12 compounds With further stepwise minor modifications to the protocol acceptable recoveries were achieved for all 12 compounds. pl 8 6 9 1 9 3 3 87 12 7 3 4 06 7 51 7 35 8 93 10 4 9 1. MW 1069 1084 1019 2180 3464 1270 4492 1296 1046 1673 1638 4118. The diverse range of therapeutic peptides detailed above were extracted from human plasma using a generic extraction. protocol the Oasis MAX and WCX sorbents and the Elution 96 well plate format With no modifications of the protocol. all extracts with the exception of BNP Enfuvirtide and Somatostatin exhibited 80 recovery and 11 matrix. effects Analysis was carried out using the PST T herapeudic Peptide Method Development Kit and ACQUITY UPLC. with Xevo TQ MS systems, P ST T hera p eut ic P e pt ide Met hod Dev elo pment Kit. T he PST T herapeutic Peptide Method Development Kit has been developed to. simplify the process of sample preparation and LC method development for. the analysis of therapeutic peptides in plasma T he kit contains an Oasis PST. Elution Method Development Plate a PST 300 C18 reversed phase column. and the detailed screening protocol which was used to generate the data shown. in this publication, In addition a comprehensive method development training seminar has been. created which describes all aspects of the method development process from the. MS conditions to the final validation of a method for the extraction of the. therapeutic peptide Desmopressin from human plasma. For more information visit www waters com pepkit,or contact your local Waters sales office. Ordering Informat ion,Description Part No Qty Box, UPLC PST T herapeutic Peptide Method Development Kit 176001835.
Oasis PST Elution Method Development Plate 186004713 1. ACQUITY UPLC BEH 300 C18 2 1 x 50 mm 1 7 m 186003685 1. 96 well 1 mL Collection Plate and Cap Mat 600001043 3. HPLC PST T herapeutic Peptide Method Development Kit 176001836. Oasis PST Elution Method Development Plate 186004713 1. XBridge BEH 300 C18 2 1 x 50 mm 3 5 m 186003607 1, 96 well 1 mL Collection Plate and Cap Mat 600001043 3. Available Waters Products Not Included in Kit,Oasis MAX 96 well Elution Plate 186001829 1. Oasis WCX 96 well Elution Plate 186002499 1,96 well 1 mL Collection Plate 186002481 50. Cap Mats for 1 mL Collection Plate 186002483 50,Disposable Reservoir Tray WAT058942 25. Extraction Manifold for 96 well Plates 186001831 1. Vacuum Box Gasket Kit includes foam top gaskets and orange O rings 186003522 2. SPE Vacuum Pump 115 V 60 Hz 725000417 1,SPE Vacuum Pump 240 V 50 Hz 725000418 1.
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Bioanalytical Method Development Strategy for Therapeutic Peptides Oasis SPE sorbents are unique in their combination of purity reproducibility stability and retention characteristics The use of the extraction selectivity of two of the Oasis mixed mode ion exchange sorbents in combination with a simple extraction protocol is a key contributor to the success rate of this generic

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