Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (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 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 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (LLE) by 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 Clozapine 1 mg/mL, from Cerilliant #C-059 , or equivalent 4.3.4 Doxepin 1 mg/mL, from Cerilliant #D-927, or equivalent 4.3.5 Desmethyldoxeopin 1 mg/mL, from Cerilliant #D-007, or equivalent 4.3.6 Fluoxetine 1 mg/mL, from Cerilliant #F-918, or equivalent 4.3.7 Norfluoxetine Oxalate 1 mg/mL, from Cerilliant #N-923, or equivalent 4.3.8 Quetiapine 1 mg/mL, from Cerilliant #Q-001, or equivalent 4.3.9 Norquetiapine 1 mg/mL, from Cerilliant #N-070, or equivalent 4.3.10 Olanzapine 1 mg/mL, from Cerilliant #O-024, or equivalent 4.3.11 Amitriptyline-D3 100 μg/mL, from Cerilliant #A-085, or equivalent 4.3.12 Nortriptyline-D3 100 μg/mL, from Cerilliant #N-902, or equivalent 4.3.13 Clozapine-D4 100 g/mL, from Cerilliant #C-091 , or equivalent 4.3.14 Doxepin-D3 100 g/mL, from Cerilliant #D-060 , or equivalent 4.3.15 N-Desmethyldoxeopin-D3 100 g/mL, from Cerilliant #D-075, or equivalent 4.3.16 Fluoxetine-D6 Oxalate 100 μg/mL, from Cerilliant #F-919, or equivalent 4.3.17 Norfluoxetine-D6 100 μg/mL, from Cerilliant #N-922, or equivalent 4.3.18 Quetiapine-D8, 100 μg/mL, from Cerilliant #Q-002, or equivalent 4.3.19 Olanzapine-D8 100 μg/mL from Cerilliant #O-035, or equivalent

Page 2 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (LLE) by LCMSMS

4.4 Working Standards and Controls

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.

4.4.1 Standard “A” (10 g/mL) A. Pipette 1 mL of the 1 mg/mL stock standards into a 100 mL volumetric flask: a. Amitriptyline b. Nortriptyline c. Clozapine d. Doxepin e. Nordoxepin f. Fluoxetine g. Norfluoxetine h. Quetiapine i. Norquetiapine j. 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 A with drug names) B. Fill the remaining volume with methanol. C. Label as Standard A. Write preparation date, expiration date, storage temperature, and initials of analyst who prepared standard.

4.4.2 Standard “B” (1.0 g/mL) A. Aliquot 2.5 mL of Standard “A” into a 25 mL volumetric flask and fill to the meniscus with methanol. B. Label as Standard “B”. 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 Standard B with drug names)

4.4.3 QC A (10 g/mL) A. Pipette 1 mL of the 1 mg/mL stock standards into a 100 mL

Page 3 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (LLE) by LCMSMS

volumetric flask: a. Amitriptyline b. Nortriptyline c. Clozapine d. Doxepin e. Nordoxepin f. Fluoxetine g. Norfluoxetine h. Quetiapine i. Norquetiapine j. 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 A 10 g/mL with drug names) B. Fill the remaining volume with methanol. C. Label as QC A 10 g/mL. Write preparation date, expiration date, storage temperature, and initials of person who prepared standard.

4.4.4 QC B (1.0 g/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 g/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 (10/1.0 g/mL) A. Aliquot 1.0/0.10 mL of each of the following standards into a 10 mL volumetric flask: a. Amitriptyline-D3 (0.10 mL) b. Nortriptyline-D3 (0.10 mL) c. Clozapine-D4 (0.10 mL) d. Doxepin-D3 (0.10 mL) e. N-Desmethyldoxepin-D3 (0.10 mL) f. Fluoxetine-D6 Oxalate (1.0 mL) g. Norfluoxetine-D6 (1.0 mL) h. Quetiapine-D8 (0.10 mL) i. Olanzapine-D8 (1.0 mL) 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.

Page 4 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (LLE) by LCMSMS

Note: The ISTDs could be prepared individually or in combination with other ISTDs.

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.

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 B 5 10 Std 2 100 B 50 10 Std 3 500 A 25 10 Std 4 1000 A 50 10 Std 5 2000 A 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

Page 5 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (LLE) by LCMSMS

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 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.

5.4 Table 2: Sample injection sequence Position Sample Number 1st Blank Mobile Phase A 2nd Blank Blood Extract 3rd Negative Control 4th Standard 1 5th Standard 2 6th Standard 3 7th Standard 4 8th Standard 5 9th Negative Control (reinjection) 10th LOW QC 12th HIGH QC 13th Blank Blood Extract (reinjection) Specimens QCs every10th injection and ending with a QC; Neg QC every 5 case injections and as needed; Blank Mobile Phase after each case

Note: MPBs are injected after each case specimen to reduce residual background signal.

Page 6 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (LLE) by LCMSMS

5.5 Instrument Parameters

5.5.1 Table 3: Mobile Phase

A 7mM Ammonium Formate + 0.05% Formic Acid B Acetonitrile

5.5.2 Table 4: LC Conditions Internal Particle Column Length Column Flow Diameter Size C18 100 mm 3 mm 3.5 µm 0.3 mL/min

HPLC Program (Gradient) Time (min.) %A %B 0 90 10 3 90 10 6.5 10 90 7.8 10 90 8 90 10 10 90 10

5.5.3 Table 5: QQQ Conditions Time segment 1 Send to waste Time segment 2 Send to MS

Precursor Product Ions Compound Collision (V) Ion (Quant/Qual) 91 30 Amitriptyline 278 105 20 233 15 Nortriptyline 264 105 20 269.9 20 Clozapine 327.1 192 44 107 20 Doxepin 280.0 235 10 107 16 Nordoxepin 266.2 202 32 44 12 Fluoxetine 310.1 148 4 30 10 Norfluoxetine 296 134 5

Page 7 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (LLE) by LCMSMS

253 20 Quetiapine 384.2 221 40 210 28 Norquetiapine 296.1 253 16 256 20 Olanzapine 313 84 24 233 12 Amitriptyline-D3 281.2 105 24 233 8 Nortriptyline-D3 267.2 105 20 272 20 Clozapine-D4 331.1 192 44 107 20 Doxepin-D3 283.0 77 55 N-Desmethyldoxepin- 107 20 269.2 D3 77 55 44 30 Fluoxetine-D6 316.5 154 4 140 4 Norfluoxetine-D6 302 30 16 258 20 Quetiapine-D8 392 226 20 261 20 Olanzapine-D8 321.2 198 44

5.5.4 Analysis of calibrators, QC’s, and case specimens by LC/MS/MS A. Perform the day of use startup of the LC/MS/MS as described in the SOP “Agilent 6460 Triple Quad System Startup and Shutdown.” B. Ensure that proper HPLC and MS conditions are established and activated. See “Agilent 6460 Data Acquisition.” C. When data acquisition is completed, process the data as per SOP “Agilent 6460 Processing Quantitative Data.” D. Make printed copies of the standards, negatives, blanks, all Qc’s, and case specimen results. E. Turn off the instrument as per SOP “Agilent 6460 Triple Quad System Startup and Shutdown.”

6.0 Data Analysis / Interpretation/ Documentation

6.1 Refer to the LCMSMS Analyses, Quality Control, Acceptance and Reporting 6.2 Drug levels are the following: 6.2.1 The calibration curves of Amitriptyline, Nortriptyline, Clozapine, Doxepin,

Page 8 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (LLE) by LCMSMS

Nordoxepin, Fluoxetine, Norfluoxetine, Quetiapine and Olanzapine are established by standard points of 10, 100, 500, 1000, and 2000 ng/mL, with QC levels at 100 and 500 ng/mL. 6.2.2 The calibration curve of Norquetiapine is established by standard points of 10, 100, 500 and 1000 ng/mL, with QC levels at 100 and 500 ng/mL.

6.3 Internal standard (ISTD) assignment 6.3.1 The following are the ISTD assignments for each analyte for data process: A. Amitriptyline - Amitriptyline-D3 B. Nortriptyline - Nortriptyline-D3 C. Clozapine - Clozapine-D4 D. Doxepin - Doxepin-D3 E. Nordoxepin - N-Desmethyldoxepin-D3 F. Fluoxetine - Fluoxetine-D6 Oxalate G. Norfluoxetine - Norfluoxetine-D6 H. Quetiapine - Quetiapine-D8 I. Norquetiapine - Quetiapine-D8 J. Olanzapine - Olanzapine-D8

6.4 LOD and LOQ 6.4.1 MPBs are injected after each case specimen to reduce residual background signal. 6.4.2 Limit of quantitation (LOQ) for all analytes is 10 ng/mL or the lowest acceptable standard on the curve. 6.4.3 Limit of detection (LOD) for all analytes is 5 ng/mL. 6.4.4 Cases results less than the LOD are reported as “None detected.”

7.0 Acceptance Criteria

7.1 Refer to the LCMSMS Analyses, Quality Control, Acceptance and Reporting Criteria SOP. 7.2 The calibration curve of Norquetiapine is allowed to use quadratic without further approval.

8.0 References

8.1 SOP: “Agilent 6460 Triple Quad System Startup and Shutdown ” 8.2 SOP: “Agilent 6460 Processing Quantitative Data” 8.3 SOP: “Agilent 6460 Data Acquisition.”

9.0 Revision History

Revision Description of Change Reviewed By Date Page 9 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: LC & LCMSMS Procedure No.: TOX.LC.3499 Title: Amitriptyline, Clozapine, Doxepin, Fluoxetine, Quetiapine and Olanzapine Rev.4 (LLE) by LCMSMS

0 Original M. Chen 4/2012 Update standard/control expiration date criteria; change to add a MPB after each case specimen; added administrative LOD; removed quadric curve acceptance criteria for norquetiapine; 1 remove to monitor for Quetiapine sulfoxide CA 1212 Added the assay for Doxepin and Nordoxepin and related information; revised the description for 0513; 2 reconstitute buffer expiration date MC 0813 Updated volumes of standard/control to prepare; added lowest acceptable standard for LOQ criteria; removed brand names or added ‘or equivalent’; removed 1 hour dry time and updated dry down temperature; removed non-critical instrument parameters. Added a split of original 0913; 3 QC into QC A and QC B. CA/JW 1113 Added Clozapine, Clozapine-D4, Doxepin-D3, N- Desmethyldoxepin-D3 and related information; Changed ISTD concentration of Fluoxetine-D6, 4 Norfluoxetine-D6 and Olanzapine-D8. MC 0114

10.0 Attachments or Appendices

10.1 Not Applicable

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