Bioanalytical Sciences Sample Preparation PDF

Bioanalytical Sciences Sample collection, handling & preparation Dr. Isabelle Kohler 1 Bioanalytical Sciences | [email protected]  Q&A INTERMEZZO SPE procedure | Extraction of MDMA and Methamphetamine (MA) in plasma I am in the lab and have received the missio...

Bioanalytical Sciences Sample collection, handling & preparation Dr. Isabelle Kohler 1 Bioanalytical Sciences | [email protected]  Q&A INTERMEZZO SPE procedure | Extraction of MDMA and Methamphetamine (MA) in plasma I am in the lab and have received the mission to analyse MA and MDMA in plasma samples using LC-MS. I need to set up a sample preparation method to extract these compounds from plasma samples. What will my sample preparation procedure look like? 2  Extraction MA and MDMA from plasma Condition Load Wash & Elute 3 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma How do I proceed?? Which information do I need? pKa of the molecules? Charge of the molecule depending on the pH? LogP of the molecule? Type of sorbent? ( which type of interactions?) Conditioning of the sorbent? What do I need to do with the plasma before loading it onto the sorbent? Washing steps? Elution? 4 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma MDMA MA pKa : 9.9 pKa : 10.2 LogP: 1.8 LogP: 2.2 Play with the ionic interactions to have a strong retention with the sorbent  Choice of sorbent? 5 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma MCX: mixed-mode strong Cation eXchange (“strong” = always charged) WCX: mixed-mode Weak Cation eXchange (“weak” = charged depending on the pH) 6 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma MCX: mixed-mode strong Cation eXchange (“strong” = always charged) WCX: mixed-mode Weak Cation eXchange (“weak” = charged depending on the pH) pH = pKa - 2 7 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Condition Load Wash & Elute 8 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Condition When you buy SPE cartridges/well-plates, the sorbent is dry. The sorbent needs to be “activated” to be sure that it interacts properly with the analytes How?  this is specified by the vendor in the manual: read it! (RTFM ) Most of the time: 1. MeOH 2. Water 9 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Load The sample needs to be pre-treated before being loaded onto the cartridge Two important aspects: 1. Is it a protein-rich matrix? If yes -> precipitate them: they will clog the sorbent. 2. Is the pH of the sample adequate for the retention of the analytes of interest? ! Flow rate during the loading step: don’t go too fast if electeostatic interactions as these are slow interactions!  max. 1 drop/s Electrostatic interactions = strong but slow interactions. 10 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Load Two important aspects: 1. Is it a protein-rich matrix? If yes -> precipitate them: they will clog the sorbent. Plasma: yes, protein-rich  do a protein precipitation Acids Organic solvents Perchloric acid Methanol Trichloroacetic acid Ethanol Acetic acid Acetonitrile m-phosphoric acid Which one? Depends on your SPE procedure! 11 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Load 12 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Load pH = pKa - 2  Load a solution at pH < ca. 7.9 13 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Load Acids Organic solvents Perchloric acid Methanol Trichloroacetic acid Ethanol Acetic acid Acetonitrile m-phosphoric acid Two important aspects: 1. Is the pH of the sample adequate for the retention of the analytes of interest?  Load a solution at pH < ca. 7.9 14 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Load Acids Perchloric acid Trichloroacetic acid Acetic acid m-phosphoric acid Selection of acid solution:  Test at least 2-3 different options 15 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Load 1. PP agent 2. plasma 5. Collection of the supernatant 6. SPE procedure 3. agitation 4. centrifugation (!! Flow rate during loading step!) 16 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Wash & Elute Acidic wash: Analytes are retained (adequate pH) Wash contaminants that are hydrophilic and not positively charged. 17 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Wash & Elute Organic wash: Analytes are retained (adequate pH) Wash contaminants that are hydrophobic and not positively charged. 18 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Wash & Elute Elution: adequate pH to break the electrostatic interactions pH = pKa + 2 Organic solvent to elute hydrophobic analytes 19 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Wash & Elute And then? Usually the last elution leads to a volume of 2-6 mL (this is an example, but depends on the sorbent size) Preconcentration factor always welcome: We will inject around 2-20 µL in LC We need around 25 µL in an LC vial Evaporation of the last elution step until dryness, and reconstitution in 25 µL (PF: 1 mL plasma to 25 µL  40x) Reconstitution solvent: usually the composition of the mobile phase at the beginning of the gradient. 20 Bioanalytical Sciences | [email protected]  Solid-phase extraction (SPE) | Choice of solvents Rule: “Like dissolves like” A polar compound has more affinity for a polar stationary than an apolar elution phase Play with the polarity of your washing and elution phases to retain or elute your compounds Know the polarity properties of your compounds  logP (logD for ionized forms) 21 Bioanalytical Sciences | [email protected]  SPE | Procedure Condition Load Wash & Elute Adjust the pH of the sample if Usually MeOH needed prior to the loading step The composition of the mixtures used for the washing and elution steps depends on The stationary phase (polarity? Charge?) The analyte(s) of interest (polarity? Charge?) !! pH of the Washing and elution steps are sample usually performed with a mixture of (adjust if organic solvent and water, with acids needed) or bases added if needed. 22 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma How do I proceed?? Which information do I need? pKa of the molecules? Charge of the molecule depending on the pH? LogP of the molecule? Type of sorbent? ( which type of interactions?) Conditioning of the sorbent? What do I need to do with the plasma before loading it onto the sorbent? Washing steps? Elution? 23 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma Condition Load Wash & Elute 24 Bioanalytical Sciences | [email protected]  Q&A INTERMEZZO SPE procedure | Extraction of MDMA and Methamphetamine (MA) in plasma 25 https://www.waters.com/waters/en_US/Waters-Oasis-Sample-Extraction-SPE-Products/nav.htm?cid=513209&locale=en_US