Nottingham Trent University – Intro Analytical Toxicology QUANTIFICATION OF CARBAMAZEPINE IN POST MORTEM BLOOD USING LC-MS TITLE: Procedure No : SOP-002 Implementation Date : February 2023 Version : 01 Replaces : New Page : 1 of 8 Copy No: Basic Liquid-Liquid Extraction (Carbamazepine): Case History: The deceased was a 56 year old woman who had a history since 1996 of self-harm via an overdose, since 1999 the deceased suffered from asthma and was prescribed uniphyllin and inhalers, in 2006 she was taken to hospital due to an overdose of paracetamol, in 2011 the deceased was admitted to hospital due to seizures, her family at the time stated she had been consuming excessive alcohol for this she was prescribed levetiracetam, in 2012 she was prescribed 112 uniphyllin tablets. On the day in the morning her husband left her alone to go to work, on the same date when her husband returned from work he witnessed the deceased lying on the sofa and appeared intoxicated he also noted that there was vomit on the sofa and the floor of the living room, the deceased denied drinking. In the evening the deceased continued to vomit and later admitted to drinking as well as taking an unknown quantity of medication. Also, in the evening the deceased got up to go to the bathroom several times and on one occasion the deceased fell and injured her nose. The next day the deceased husband phone his work and said that the deceased had tried to commit suicide and later that day the brother of the deceased investigated the deceased house and saw her lying on the sofa when her husband could not wake her up an ambulance was called who later found the deceased lying on the living room floor. You are required to analyse a blood sample from the deceased for carbamazepine and provide your opinion on the possible role of this drug in the death • • • • COSHH ASSESSMENT Carbamazepine – is a toxic and carcinogenic chemical that can cause vomiting, dizziness, swollen tongue and loss of balance. Brompheniramine – is a toxic and irritant chemical that can cause agitation, comas, seizures, delirium, drowsiness, fever and irregular or rapid heartbeat. Hexane – is a flammable, toxic and carcinogenic chemical that can cause severe lung irritation, coughing, pulmonary oedema; excitement followed by depression. Ingestion causes nausea, vomiting, swelling of abdomen, headache, depression. Ethyl acetate – is a flammable and toxic chemical and can cause irritation of the eyes, nose and throat, followed by headache, nausea, vomiting, sleepiness, and unconsciousness. High concentrations can cause CNS depression and congestion of the liver and kidneys. Written by: Chinyere Williams Date : 01/02/2023 Approved by : Date : Nottingham Trent University – Intro Analytical Toxicology QUANTIFICATION OF CARBAMAZEPINE IN POST MORTEM BLOOD USING LC-MS TITLE: Procedure No : SOP-002 Implementation Date : February 2023 Version : 01 Replaces : New Page : 2 of 8 Copy No: PUBLISHED METHODS Environmental exposure to pharmaceuticals: A new technique for trace analysis of carbamazepine and its metabolites in human urine – Ammonium acetate used https://www.sciencedirect.com/science/article/pii/S0269749116301324 Liquid chromatography–tandem mass spectrometry for the analysis of pharmaceutical residues in environmental samples: a review – Formic acid used https://www.sciencedirect.com/science/article/pii/S0021967304021648 INTRODUCTION Using a basic liquid-liquid extraction, carbamazepine is extracted from post mortem blood samples by making the samples slightly basic and extracting with a Hexane: Ethyl Acetate Solution. An aliquot of the extract is analysed by Liquid Chromatography Mass Spectrometry (LC/MS). OPERATING PROCEDURE 1.0 SPECIMEN REQUIREMENTS. Postmortem blood 2.0 REAGENTS AND STANDARDS a. b. c. d. e. f. g. h. Carbamazepine, 1 mg/mL Paracetamol, 1 mg/mL De-ionised Water Hexane: Ethyl Acetate Solution (1:9) 0.2M Sodium Carbonate (aq) Methanol Formic Acid Blank horse blood Written by: Chinyere Williams Date : 01/02/2023 Approved by : Date : Nottingham Trent University – Intro Analytical Toxicology QUANTIFICATION OF CARBAMAZEPINE IN POST MORTEM BLOOD USING LC-MS TITLE: Procedure No : SOP-002 Implementation Date : February 2023 Version : 01 Replaces : New Page : 3 of 8 Copy No: 3.0 CONSUMABLES a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p. 1 x 100mL glass jar – to store the Hexane:Ethyl Acetate (1:9) 1 x 10mL volumetric flask – to make up the initial internal standard 1 x 20mL plastic tube – to store the blank horse blood 1 x 50mL plastic tube – to store the methanol 3 x 500mL Duran bottle – make up mobile phase A and B 5 x plastic flat bottom tube – make up QC solutions, make up Carbamazepine standard solution, make up internal calibration solution and the methanol/water (5:5) mix Dram tubes – To dry out the upper layer organic solvent Eppendorf microtubes – make up calibration curve solutions and QC solutions HPLC tubes – to put all solutions that need analysing in Weighing boats Weighing scales Automatic pipettes Spatula Centrifuge Drying station LC-MS 4.0 SOLUTIONS, INTERNAL STANDARDS, CALIBRATORS AND CONTROLS a. 1 M Sodium Carbonate in water – in a 100mL volumetric flask [Prepared by Technical Staff]. Provide calculations to make this solution: Written by: Chinyere Williams Date : 01/02/2023 Approved by : Date : Nottingham Trent University – Intro Analytical Toxicology QUANTIFICATION OF CARBAMAZEPINE IN POST MORTEM BLOOD USING LC-MS TITLE: Procedure No : SOP-002 Implementation Date : February 2023 Version : 01 Replaces : New Page : 4 of 8 Copy No: b. 0.2 M Sodium Carbonate in water – in a 100mL volumetric [Prepared by Technical Staff]. Provide calculations to make this solution: c. Mobile Phase A – Measure out 350mL of Methanol and add to a 500mL Duran bottle. Measure out 137.5mL of deionised water and add to the Duran bottle. Finally add 12.5mL of Formic Acid to the bottle. Mix vigorously and sonicate for 15 minutes before use [Prepared by Technical Staff]. d. Mobile Phase B- Measure out 350mL of Methanol and add to a 500mL Duran bottle. Measure 150mL of deionised water and add to bottle. Mix vigorously and sonicate for 15 minutes before use [Prepared by Technical Staff]. e. Carbamazepine Stock Solution [Prepared by Technical Staff] i. Prepare a 1mg/mL a Carbamazepine stock solution. If 1283 µg of carbamazepine was weighed, how much solvent is needed to make stock solution? Written by: Chinyere Williams Date : 01/02/2023 Approved by : Date : Nottingham Trent University – Intro Analytical Toxicology QUANTIFICATION OF CARBAMAZEPINE IN POST MORTEM BLOOD USING LC-MS TITLE: Procedure No : SOP-002 Implementation Date : February 2023 Version : 01 Replaces : New Page : 5 of 8 Prepare your calibrators and QCs (show your work) SOLUTION SOLUTION VOLUME OF VOLUME OF ID USED SOLUTION USED BLANK HORSE (ML) BLOOD (ML) CAL 1 Calibration 1 Stock Solution CAL 2 CAL 1 0.5 CAL 3 CAL 2 0.5 CAL 4 CAL 3 0.5 CAL 5 CAL 4 0.5 QUALITY CONTROL SAMPLES HQC Stock Solution 1 LQC ? 1 Copy No: FINAL CONCENTRATION OF CARBAMAZEPINE (MG/L) 30 15 7.5 3.75 1.875 20 5 f. Internal Standard Stock Solution. i. Prepare a 1mg/mL paracetamol solution. If using the salt form, determine the amount of salt need to equal 1mg of the free base and weigh this amount. Stock solutions are stored capped in the refrigerator. ππππππππβππππ π΄π΄π΄π΄π΄π΄π΄π΄π΄π΄π΄π΄ ππππ π·π·π·π·π·π·π·π· ππππππππππππππππ (ππππ) οΏ½ οΏ½ πΉπΉπΉπΉ π·π·π·π·π·π·π·π· ππππππππ οΏ½ οΏ½ πΉπΉπΉπΉ π·π·π·π·π·π·π·π· π΅π΅π΅π΅π΅π΅π΅π΅ ππππ πΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆπΆ ππππ π π π π π π π π π π π π π π π π ππππππππππππππππ ( ππππ ) 1 οΏ½ 302.84 οΏ½ οΏ½266.381οΏ½ 1 ππππππππππππ ππππ π π π π π π π π π π π π π π ππππππππππππππππ = 1.136mL Is this correct (Prove your answer)? ii. Internal Standard Working Solution- Pipette 5mL of 0.2M Sodium Carbonate (aq) into a 10mL volumetric. Add 100µL of internal standard Dilution solution to the volumetric and then make up to volume with 0.2M Sodium Carbonate (aq). Mix thoroughly. Written by: Chinyere Williams Date : 01/02/2023 Approved by : Date : Nottingham Trent University – Intro Analytical Toxicology QUANTIFICATION OF CARBAMAZEPINE IN POST MORTEM BLOOD USING LC-MS TITLE: Procedure No : SOP-002 Implementation Date : February 2023 Version : 01 Replaces : New Page : 6 of 8 Copy No: 5.0 APPARATUS a. b. c. d. e. f. g. h. i. Test Tubes, 16 x 12.5 mm, conical bottom, polypropylene with polypropylene caps. Centrifuge capable of 2000- 3000 rpm 4ml glass dram vials and lids. Plastic pipettes. Multishaker Evaporator/ Concentrator. Vortex Mixer HPLC Autosampler vials and caps HPLC-DAD: Dionex Model: i. HPLC Instrument Conditions. The following instrument conditions may be modified to adjust or improve separation and sensitivity. 1. Elution Conditions. a. Column: Restex Raptor; C18 2.7µm, 100 x 2.1mm b. Guard Column: N/A c. Column Thermostat: ca 25C d. Mobile Phase A: - 60% e. Mobile Phase B: - 40%. f. Initial Flow Rate: 0.3mL/min g. Injection Volume: 1µL h. Run time: 12min i. Gradient: Isocratic j. Single Ion 152nm and 237nm k. Retention Times: Carbamazepine ca 7.76min l. Paracetamol: ca 0.89min Written by: Chinyere Williams Date : 01/02/2023 Approved by : Date : Nottingham Trent University – Intro Analytical Toxicology QUANTIFICATION OF CARBAMAZEPINE IN POST MORTEM BLOOD USING LC-MS TITLE: Procedure No : SOP-002 Implementation Date : February 2023 Version : 01 Replaces : New Page : 7 of 8 Copy No: 6.0 PROCEDURE a. Label microtubes appropriately with calibrators, quality controls and case samples IDs. Prepare these tubes in duplicate. b. Add 0.2mL calibration standard, quality control samples or case blood into the appropriately labelled test tube. c. Add 0.15mL of Internal Standard Working Solution to each tube. Vortex briefly. d. Add 1.5mL of Hexane: Ethyl Acetate to each microtube, cap, vortex and place on shaker for 5 minutes. e. Centrifuge for 5mins at ca 2500rpm. f. Transfer upper solvent layer into a clean appropriately labelled dram vial using a clean disposable Pasteur pipette. g. Evaporate to dryness at 60°C under air. h. Reconstitute in 100 µL of methanol. i. Transfer 100 µL of the reconstituted solution to a clean labelled HPLC vial. j. Inject 1µL onto LCMS. The space below is provided for extra notes and details Written by: Chinyere Williams Date : 01/02/2023 Approved by : Date : Nottingham Trent University – Intro Analytical Toxicology TITLE: QUANTIFICATION OF CARBAMAZEPINE IN POST MORTEM BLOOD USING LC-MS Procedure No : SOP-002 Implementation Date : February 2023 Version : 01 Replaces : New Page : 8 of 8 Copy No:
