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Toxicology Approved By Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Doxepin, Fluoxetine, Quetiapine and Olanzapine (LLE) by Rev.3 LCMSMS 1.0 Purpose 1.1 Amitriptyline, Nortriptyline, Doxepin, Nordoxepin (Desmethyldoxepin), Fluoxetine, Norfluoxetine, Quetiapine, Norquetiapine, and Olanzapine are assayed by High Performance Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) using multiple reaction monitoring (MRM) and their corresponding deuterated standards. 2.0 Scope 2.1 The analysis is applicable to all types of forensic specimens when compared with matrix matched controls. 3.0 Definitions and Abbreviations 3.1 None Applicable 4.0 Materials 4.1 Instruments and Equipment 4.1.1 Pipettes 4.1.2 13 x 100 mm screw cap test tubes 4.1.3 16 x 100 mm test tubes 4.1.4 Centrifuge 4.1.5 Autosampler vials 4.1.6 Vial caps with puncture seals 4.1.7 Solvent evaporator 4.1.8 Nitrogen compressed gas 4.1.9 0.2 m Filters 4.1.10 C18 PLC column 4.1.11 LC-MS/MS 4.1.12 3 c.c. Syringe 4.2 Reagents 4.2.1 Extraction Solvent: 1-Chlorobutane 4.2.2 Acetonitrile, HPLC grade 4.2.3 Formic Acid 88% 4.2.4 Water, 18.2 mega ohm or HPLC grade 4.2.5 Ammonium formate. 4.2.6 Saturated Sodium Borate Buffer A. Weigh 100 g of Sodium Tetraborate Page 1 of 9 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Doxepin, Fluoxetine, Quetiapine and Olanzapine (LLE) by Rev.3 LCMSMS B. Add to 900 mL of deionized water. C. Heat with stirring bar in a 1 L beaker until dissolved. D. Add 50 mL of 10 N Sodium Hydroxide E. Stir and allow to cool. F. Check pH with meter. Should be pH 12 + 0.2. Adjust as necessary with 10 N NaOH. G. Stable 6 months at room temperature. 4.2.7 Mobile phase A: 0.05% formic Acid and 7 mM ammonium formate A. Measure 1L of Milli-Q filtered or HPLC grade water into a clean 1 L bottle. B. Add 0.45 g of ammonium formate and 0.568 mL of formic acid to the water. Mix well. C. Stable for 3 months at room temperature. 4.2.8 Mobile phase B: Acetonitrile. Stable for 3 months at room temperature. 4.2.9 Reconstitution buffer: 90% of 0.05% formic acid 7mM ammonium formate in water / 10% acetonitrile. Expiration date is date mobile phase used. 4.3 Stock Standards and Controls Note: Cerilliant standards should be used within the manufacturer’s expiration date and should be stored at the recommended storage temperature. 4.3.1 Amitriptyline 1 mg/mL, from Cerilliant #A-923, or equivalent 4.3.2 Nortriptyline 1 mg/mL , from Cerilliant #N-907, or equivalent 4.3.3 Doxepin 1 mg/mL, from Cerilliant #D-927, or equivalent 4.3.4 Desmethyldoxeopin 1 mg/mL, from Cerilliant #D-007, or equivalent 4.3.5 Fluoxetine 1 mg/mL, from Cerilliant #F-918, or equivalent 4.3.6 Norfluoxetine Oxalate 1 mg/mL, from Cerilliant #N-923, or equivalent 4.3.7 Quetiapine 1 mg/mL, from Cerilliant #Q-001, or equivalent 4.3.8 Norquetiapine 1 mg/mL, from Cerilliant #N-070, or equivalent 4.3.9 Olanzapine 1 mg/mL, from Cerilliant #O-024, or equivalent 4.3.10 Amitriptyline-D3 100 μg/mL, from Cerilliant #A-085, or equivalent 4.3.11 Nortriptyline-D3 100 μg/mL, from Cerilliant #N-902, or equivalent 4.3.12 Fluoxetine-D6 Oxalate 100 μg/mL, from Cerilliant #F-919, or equivalent 4.3.13 Norfluoxetine-D6 100 μg/mL, from Cerilliant #N-922, or equivalent 4.3.14 Quetiapine-D8, 100 μg/mL, from Cerilliant #Q-002, or equivalent 4.3.15 Olanzapine-D8 100 μg/mL from Cerilliant #O-035, or equivalent 4.4 Working Standards and Controls Note: Standards and controls must be prepared by different analysts. Page 2 of 9 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Doxepin, Fluoxetine, Quetiapine and Olanzapine (LLE) by Rev.3 LCMSMS Note: Prepared working internal standards/standards/controls may not have an expiration date that exceeds the expiration date of the stock standard used to prepare it. Note: Prepared working internal standards/standards/controls are stable until the manufacturer’s expiration date when stored at manufacturer’s recommended storage temperature. 4.4.1 Standard “B” (10 ug/mL) A. Pipette 1 mL of the 1 mg/mL stock standards into a 100 mL volumetric flask: a. Amitriptyline b. Nortriptyline c. Doxepin d. Nordoxepin e. Fluoxetine f. Norfluoxetine g. Quetiapine h. Norquetiapine i. Olanzapine (Do not assume that the ampoule contains exactly 1 mL, so transfer using a pipette.) (Analytes can be prepared in the same vial or individually in vials labeled as STD B with drug names) B. Fill the remaining volume with methanol. C. Label as Standard B. Write preparation date, expiration date, storage temperature, and initials of analyst who prepared standard. 4.4.2 Standard “C” (1.0 ug/mL) A. Aliquot 2.5 mL of Standard “B” into a 25 mL volumetric flask and fill to the meniscus with methanol. B. Label as Standard “C”. Write preparation date, expiration date, storage temperature, and initials of analyst preparing standard. (Analytes can be prepared in the same vial or individually in vials labeled as STD C with drug names) 4.4.3 QC A (10 ug/mL) A. Pipette 1 mL of the 1 mg/mL stock standards into a 100 mL volumetric flask: a. Amitriptyline b. Nortriptyline c. Doxepin d. Nordoxepin Page 3 of 9 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Doxepin, Fluoxetine, Quetiapine and Olanzapine (LLE) by Rev.3 LCMSMS e. Fluoxetine f. Norfluoxetine g. Quetiapine h. Norquetiapine i. Olanzapine (Do not assume that the ampoule contains exactly 1 mL, so transfer using a pipette.) (Analytes can be prepared in the same vial or individually in vials labeled as QC 10 ug/mL with drug names) B. Fill the remaining volume with methanol. C. Label as QC A 10 ug/mL. Write preparation date, expiration date, storage temperature, and initials of person who prepared standard. 4.4.4 QC B (1.0 ug/mL) A. Pipette 1 mL of QC A into a 10 mL volumetric flask. B. Fill the remaining volume with methanol. C. Label as QC B 1.0 ug/mL. Write preparation date, expiration date, storage temperature, and initials of person who prepared standard. Note: The analytes could be prepared individually or in combination with other analytes. 4.4.5 Internal Standard Mix (1 ug/mL) A. Aliquot 1 mL of each of the following standards into a 100 mL volumetric flask: a. Amitriptyline-D3 b. Nortriptyline-D3 c. Fluoxetine-D6 Oxalate d. Norfluoxetine-D6 e. Quetiapine-D8 f. Olanzapine-D8 B. Fill up the flask to the meniscus with HPLC grade methanol. C. Label as Internal Standard. Write preparation date, expiration date, storage temperature and initials of person preparing standard. 5.0 Extraction Procedure 5.1 Standards and Controls 5.1.1 Label a sufficient number of 13 x 100 mm screw cap tubes for curve, QCs and case specimens. 5.1.2 Add 10 µL of working internal standard to every tube except for the reagent blank tube. Page 4 of 9 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Doxepin, Fluoxetine, Quetiapine and Olanzapine (LLE) by Rev.3 LCMSMS 5.1.3 Add the indicated volumes of Working Standards or Controls. An additional negative control should be extracted for every 5 case specimens extracted and alternating low or high QCs extracted for every 10 extracted specimens. 5.1.4 Table 1: Pipette volumes Tube Target STD Aliquot I.S. Conc. stock stock (ng/mL) (µL) (µL) Reagent blank ---- ---- ---- ---- Neg Control ---- ---- ---- 10 Std 1 10 C 5 10 Std 2 100 C 50 10 Std 3 500 B 25 10 Std 4 1000 B 50 10 Std 5 2000 B 100 10 Low QC 100 QC B 50 10 High QC 500 QC A 25 10 5.2 Sample Preparation 5.2.1 Pipette 0.5 mL of negative blood for respective curve sample and QCs into the labeled 13 X 100 mm screw cap tubes, use other appropriate matrix QC controls if needed. 5.2.2 Pipette 0.5 mL of case specimens into respective tubes. 5.2.3 Add 0.2 mL of saturated borate buffer 5.2.4 Add 3 mL of 1-Chlorobutane 5.2.5 Cap tubes, vortex for 5 seconds, centrifuge for 5 minutes at 3000 rpm. 5.2.6 Transfer the clear organic solvent layer into a labeled, clean 16x100 mm tube. 5.2.7 Evaporate solvent from tubes with compressed nitrogen gas at ≤37°C 5.3 Sample Reconstitution Page 5 of 9 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Doxepin, Fluoxetine, Quetiapine and Olanzapine (LLE) by Rev.3 LCMSMS 5.3.1 Reconstitute dried tubes with 2 mL of: 90% of 0.05% formic acid 7mM ammonium formate in water / 10% acetonitrile. 5.3.2 Filter each sample through 0.2 m syringe filters with 3 cc syringes into autosampler vials and cap.
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