TOX.LC.3499 Rev 07, Effective 2015-08-12 to Present
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Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.7 (LLE) by LCMSMS 1.0 Purpose 1.1 Amitriptyline, Nortriptyline, Clozapine, 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 Vortex mixer 4.1.5 Centrifuge 4.1.6 Autosampler vials 4.1.7 Vial caps with puncture seals 4.1.8 Solvent evaporator 4.1.9 Nitrogen compressed gas 4.1.10 0.2 m Filters 4.1.11 3 c.c. Syringe 4.1.12 C18 PLC column 4.1.13 LC-MS/MS 4.2 Reagents 4.2.1 Methanol, HPLC grade 4.2.2 n-Butyl Chloride, HPLC grade 4.2.3 Acetonitrile, HPLC grade 4.2.4 Formic Acid 88% 4.2.5 Water, 18.2 mega ohm or HPLC grade 4.2.6 Ammonium formate. Page 1 of 12 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.7 (LLE) by LCMSMS 4.2.7 Saturated Sodium Borate Buffer A. Weigh 100 g of Sodium Tetraborate 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. 4.2.8 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. 4.2.9 Mobile phase B: Acetonitrile. Stable for 3 months at room temperature. 4.2.10 Reconstitution buffer: 90% of 0.05% formic acid 7mM ammonium formate in water / 10% acetonitrile. A. Prepare enough for 2 mL per sample B. For every 9 mL of 0.05% formic acid + 7mM ammonium formate in water add 1 mL of acetonitrile and mix. C. Stable for 3 months at room temperature (not to exceed expiration date of 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, Cerilliant #A-923, or equivalent 4.3.2 Nortriptyline 1 mg/mL, Cerilliant #N-907, or equivalent 4.3.3 Clozapine 1 mg/mL, Cerilliant #C-059 , or equivalent 4.3.4 Doxepin 1 mg/mL, Cerilliant #D-927, or equivalent 4.3.5 Desmethyldoxepin 1 mg/mL, Cerilliant #D-007, or equivalent 4.3.6 Fluoxetine 1 mg/mL, Cerilliant #F-918, or equivalent 4.3.7 Norfluoxetine Oxalate 1 mg/mL, Cerilliant #N-923, or equivalent 4.3.8 Quetiapine 1 mg/mL, Cerilliant #Q-001, or equivalent 4.3.9 Norquetiapine 1 mg/mL, Cerilliant #N-070, or equivalent 4.3.10 Olanzapine 1 mg/mL, Cerilliant #O-024, or equivalent 4.3.11 Amitriptyline-D3 100 μg/mL, Cerilliant #A-085, or equivalent 4.3.12 Nortriptyline-D3 100 μg/mL, Cerilliant #N-902, or equivalent 4.3.13 Clozapine-D4 100 g/mL, Cerilliant #C-091 , or equivalent 4.3.14 Doxepin-D3 100 g/mL, Cerilliant #D-060 , or equivalent Page 2 of 12 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.7 (LLE) by LCMSMS 4.3.15 N-Desmethyldoxepin-D3 100 g/mL, Cerilliant #D-075, or equivalent 4.3.16 Fluoxetine-D6 Oxalate 100 μg/mL, Cerilliant #F-919, or equivalent 4.3.17 Norfluoxetine-D6 100 μg/mL, Cerilliant #N-922, or equivalent 4.3.18 Quetiapine-D8, 100 μg/mL, Cerilliant #Q-002, or equivalent 4.3.19 Olanzapine-D8 100 μg/mL Cerilliant #O-035, or equivalent 4.4 Working Standards Note: Standards and controls must be prepared by different analysts. 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 (<0°C). 4.4.1 Amitriptyline/Nortriptyline Working Standard A 10 µg/mL A. Pipette 250 µL of amitriptyline 1.0 mg/mL stock standard into a 25 mL volumetric flask B. Pipette 250 µL of nortriptyline 1.0 mg/mL stock standard into the same 25 mL volumetric flask C. Dilute to 25 mL with methanol 4.4.2 Amitriptyline/Nortriptyline Working Standard B 1.0 µg/mL A. Pipette 2.5 mL of Amitriptyline/Nortriptyline Standard A into a 25 mL volumetric flask. B. Dilute to 25 mL with methanol. 4.4.3 Clozapine Working Standard A 10 µg/mL A. Pipette 250 µL of clozapine 1.0 mg/mL stock standard into a 25 mL volumetric flask. B. Dilute to 25 mL with methanol. 4.4.4 Clozapine Working Standard B 1.0 µg/mL A. Pipette 2.5 mL of Clozapine Standard A into a 25 mL volumetric flask B. Dilute to 25 mL with methanol. 4.4.5 Doxepin/Nordoxepin Working Standard A 10 µg/mL A. Pipette 250 µL of doxepin 1.0 mg/mL stock standard into a 25 mL volumetric flask B. Pipette 250 µL of desmethyldoxepin 1.0 mg/mL stock standard into the same 25 mL volumetric flask Page 3 of 12 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.7 (LLE) by LCMSMS C. Dilute to 25 mL with methanol 4.4.6 Doxepin/Nordoxepin Working Standard B 1.0 µg/mL A. Pipette 2.5 mL of Doxepin/Nordoxepin Standard A into a 25 mL volumetric flask B. Dilute to 25 mL with methanol 4.4.7 Fluoxetine/Norfluoxetine Working Standard A 10 µg/mL A. Pipette 250 µL of fluoxetine 1.0 mg/mL stock standard into a 25 mL volumetric flask B. Pipette 250 µL of norfluoxetine 1.0 mg/mL stock standard into the same 25 mL volumetric flask C. Dilute to 25 mL with methanol 4.4.8 Fluoxetine/Norfluoxetine Working Standard B 1.0 µg/mL A. Pipette 2.5 mL of Fluoxetine/Norfluoxetine Standard A into a 25 mL volumetric flask B. Dilute to 25 mL with methanol 4.4.9 Olanzapine Working Standard A 10 µg/mL A. Pipette 250 µL of olanzapine 1.0 mg/mL stock standard into a 25 mL volumetric flask B. Dilute to 25 mL with methanol 4.4.10 Olanzapine Working Standard B 1.0 µg/mL A. Pipette 2.5 mL of Olanzapine Standard A into a 25 mL volumetric flask B. Dilute to 25 mL with methanol 4.4.11 Quetiapine/Norquetiapine Working Standard A 10 µg/mL A. Pipette 250 µL of quetiapine 1.0 mg/mL stock standard into a 25 mL volumetric flask B. Pipette 250 µL of norquetiapine 1.0 mg/mL stock standard into the same 25 mL volumetric flask C. Dilute to 25 mL with methanol 4.4.12 Quetiapine/Norquetiapine Working Standard B 1.0 µg/mL A. Pipette 2.5 mL of Quetiapine/Norquetiapine Standard A into a 25 mL volumetric flask B. Dilute to 25 mL with methanol 4.5 Working Controls Page 4 of 12 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.7 (LLE) by LCMSMS 4.5.1 Amitriptyline/Nortriptyline Working QC A 10 µg/mL A. Pipette 250 µL of amitriptyline 1.0 mg/mL stock control into a 25 mL volumetric flask B. Pipette 250 µL of nortriptyline 1.0 mg/mL stock control into the same 25 mL volumetric flask C. Dilute to 25 mL with methanol 4.5.2 Amitriptyline/Nortriptyline Working QC B 1.0 µg/mL A. Pipette 2.5 mL of Amitriptyline/Nortriptyline QC A into a 25 mL volumetric flask. B. Dilute to 25 mL with methanol 4.5.3 Clozapine Working QC A 10 µg/mL A. Pipette 250 µL of clozapine 1.0 mg/mL stock control into a 25 mL volumetric flask B. Dilute to 25 mL with methanol 4.5.4 Clozapine Working QC B 1.0 µg/mL A. Pipette 2.5 mL of Clozapine QC A into a 25 mL volumetric flask. B. Dilute to 25 mL with methanol 4.5.5 Doxepin/Nordoxepin Working QC A 10 µg/mL A.