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. Pipette 250 µL of doxepin 1.0 mg/mL stock control into a 25 mL volumetric flask B. Pipette 250 µL of desmethyldoxepin 1.0 mg/mL stock control into the same 25 mL volumetric flask C. Dilute to 25 mL with methanol

4.5.6 Doxepin/Nordoxepin Working QC B 1.0 µg/mL A. Pipette 2.5 mL of Doxepin/Nordoxepin QC A into a 25 mL volumetric flask. B. Dilute to 25 mL with methanol

4.5.7 Fluoxetine/Norfluoxetine Working QC A 10 µg/mL A. Pipette 250 µL of fluoxetine 1.0 mg/mL stock control into a 25 mL volumetric flask B. Pipette 250 µL of norfluoxetine 1.0 mg/mL stock control into the same 25 mL volumetric flask C. Dilute to 25 mL with methanol

4.5.8 Fluoxetine/Norfluoxetine Working QC B 1.0 µg/mL A. Pipette 2.5 mL of Fluoxetine/Norfluoxetine QC A into a 25 mL volumetric flask.

Page 5 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

B. Dilute to 25 mL with methanol

4.5.9 Olanzapine Working QC A 10 µg/mL A. Pipette 250 µL of olanzapine 1.0 mg/mL stock control into a 25 mL volumetric flask B. Dilute to 25 mL with methanol

4.5.10 Olanzapine Working QC B 1.0 µg/mL A. Pipette 2.5 mL of Olanzapine QC A into a 25 mL volumetric flask. B. Dilute to 25 mL with methanol

4.5.11 Quetiapine/Norquetiapine Working QC A 10 µg/mL A. Pipette 250 µL of quetiapine 1.0 mg/mL stock control into a 25 mL volumetric flask B. Pipette 250 µL of norquetiapine 1.0 mg/mL stock control into the same 25 mL volumetric flask C. Dilute to 25 mL with methanol

4.5.12 Quetiapine/Norquetiapine Working QC B 1.0 µg/mL A. Pipette 2.5 mL of Quetiapine/Norquetiapine QC A into a 25 mL volumetric flask. B. Dilute to 25 mL with methanol

4.6 Working Internal Standards (ISTD)

4.6.1 Amitriptyline-D3/Nortriptyline-D3 Working ISTD 1.0 µg/mL A. Pipette 100 µL of amitriptyline-D3 100 µg/mL stock internal standard into a 10 mL volumetric flask B. Pipette 100 µL of nortriptyline-D3 100 µg/mL stock internal standard into the same 10 mL volumetric flask C. Dilute to 10 mL with methanol

4.6.2 Clozapine-D4 Working ISTD 1.0 µg/mL A. Pipette 100 µL of clozapine-D4 100 g/mL stock internal standard into a 10 mL volumetric flask B. Dilute to 10 mL with methanol

4.6.3 Doxepin-D3/Nordoxepin-D3 Working ISTD 1.0 µg/mL A. Pipette 100 µL of doxepin-D3 100 g/mL stock internal standard into a 10 mL volumetric flask B. Pipette 100 µL of desmethyldoxepine-D3 100 g/mL stock internal standard into the same 10 mL volumetric flask C. Dilute to 10 mL with methanol

Page 6 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

4.6.4 Fluoxetine-D6/Norfluoxetine-D6 Working ISTD 10 µg/mL A. Pipette 1.0 mL of fluoxetine-D6 100 μg/mL stock internal standard into a 10 mL volumetric flask B. Pipette 1.0 mL of norfluoxetine-D6 100 μg/mL stock internal standard into the same 10 mL volumetric flask C. Dilute to 10 mL with methanol

4.6.5 Olanzapine-D8 Working ISTD 10 µg/mL A. Pipette 1.0 mL of olanzapine-D8 100 μg/mL stock internal standard into a 10 mL volumetric flask B. Dilute to 10 mL with methanol

4.6.6 Quetiapine-D8 Working ISTD 1.0 µg/mL A. Pipette 100 µL of quetiapine-D8 100 µg/mL stock internal standard into a 10 mL volumetric flask B. Dilute to 10 mL with methanol

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.

Table 1: Tube Target STD Aliquot I.S. Conc. stock stock (ng/mL) (µL) (µL) Reagent blank ------Negative 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

Page 7 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

5.2 Sample Preparation

5.2.1 Pipette 0.5 mL of negative matrix for respective curve sample and QCs into the labeled 13 X 100 mm screw cap tubes

5.2.2 Pipette 0.5 mL of case specimens into respective tubes.

5.2.3 Add 0.2 mL of pH 12 saturated borate buffer.

5.2.4 Add 3 mL of n-butyl chloride.

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.

5.2.8 Reconstitute with 2 mL of reconstitution buffer (90:10 0.05% FA+ 7mM AF H2O : ACN).

5.2.9 Filter each sample through 0.2µm syringe filters with 3 cc syringes into autosampler vials and cap.

5.3 Table 2: Sample injection sequence Position Number Sample 1 Blank Mobile Phase 2 Reagent Blank 3 Negative Control 4 Standard 1 5 Standard 2 6 Standard 3 7 Standard 4 8 Standard 5 3 Negative Control (reinjection) 9 LOW QC 10 HIGH QC 2 Reagent Blank (reinjection) 11 Specimens

Page 8 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

QCs every 10th injection and ending with a QC; Neg QC every 5 case injections and as needed; Mobile Phase Blank after each liver, urine and stomach contents specimen.

5.4 Instrument Parameters

5.4.1 Table 3: Mobile Phase A 7mM Ammonium Formate + 0.05% Formic Acid B Acetonitrile

5.4.2 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

5.4.3 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 327.0 192 44 107 20 Doxepin 280.0 235 10 107 16 Nordoxepin 266.2 202 32 Fluoxetine 310.1 44 12

Page 9 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

148 4 30 10 Norfluoxetine 296 134 5 253 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

6.0 Data Analysis / Interpretation/ Documentation

6.1 Refer to SOP: TOX.LC.3488 “Agilent 6460 Triple Quad Processing Quantitative Data”

6.2 Drug levels are the following: 6.2.1 The calibration curves of Amitriptyline, Nortriptyline, Clozapine, Doxepin, 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 with concentrations:

Page 10 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

6.3.1 The following are the ISTD assignments for each analyte: A. Amitriptyline - Amitriptyline-D3 (20 ng/mL) B. Nortriptyline - Nortriptyline-D3 (20 ng/mL) C. Clozapine - Clozapine-D4 (20 ng/mL) D. Doxepin - Doxepin-D3 (20 ng/mL) E. Nordoxepin - N-Desmethyldoxepin-D3 (20 ng/mL) F. Fluoxetine - Fluoxetine-D6 Oxalate (200 ng/mL) G. Norfluoxetine - Norfluoxetine-D6 (200 ng/mL) H. Quetiapine - Quetiapine-D8 (20 ng/mL) I. Norquetiapine - Quetiapine-D8 (20 ng/mL) J. Olanzapine - Olanzapine-D8 (200 ng/mL)

6.4 LOD and LOQ 6.4.1 Limit of quantitation (LOQ) for all analytes is 10 ng/mL or the lowest acceptable standard on the curve. 6.4.2 Limit of detection (LOD) for all analytes is 5 ng/mL. 6.4.3 Cases results less than the LOD are reported as “None detected.”

6.5 Additional specimen testing 6.5.1 If blood amitriptyline concentration exceeds 0.50 mg/L, request liver quantitation testing if liver is available.

7.0 Acceptance Criteria

7.1 Refer to the LCMSMS Analyses, Quality Control, Acceptance and Reporting Criteria SOP.

7.2 Report norquetiapine results qualitatively.

8.0 References

8.1 SOP: TOX09.3470 “Agilent 6460 Triple Quad System Startup and Shutdown” 8.2 SOP: TOX.LC.3484 “Agilent 6460 LC/Triple Quad Daily Maintenance, Tuning, and Calibration” 8.3 SOP: TOX.09.3469 “Agilent 6460 Triple Quad Data Acquisition” 8.4 SOP: TOX07.3409 “LC/MS/MS Chromatographic Peak Check”

9.0 Revision History

Revision Description of Change Reviewed By Date Added the assay for Doxepin and Nordoxepin and related information; revised the description for reconstitute buffer 0513; 2 expiration date MC 0813 Page 11 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

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, Norfluoxetine-D6 4 and Olanzapine-D8. MC 0114 Correct clozapine ion; remove norquetiapine quadratic acceptance criteria; report norquetiapine results 0215 5 qualitatively CA 0415 Prepare std/qc separately; add ISTD final concentration; change requirement for mobile phase blanks between all 6 specimens to liver, urine and stomach content specimens CA/T. Gray 0616 Add amitriptyline liver request if blood concentration 7 exceeds 0.50 mg/L T. Gray 0816

10.0 Attachments or Appendices

10.1 Not Applicable

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