Arkansas Department of Health Public Health Laboratory

Environmental Chemistry Section Chemical Terrorism Laboratory

Standard Operating Procedure

for

Chiral Analysis of Synthetic Cannabinoids: AM2201 and JWH-018 and their Metabolites

in Urine by LC-MS/MS

December 18, 2012

Approved by: Laboratory Supervisor Date Chemical Terrorism Lab Katie Seely, Ph.D. Section Director Date Environmental Sciences Jeffery Moran, Ph.D. Section Director Date Quality Assurance Pat Darsey Director Date Public Health Laboratory Glen Baker, M.D.

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Analyst Signature Page

By signing below, I acknowledge that I have read and fully understand this document. Name (print) Name (Signature) Date

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Table of Contents

1.0 Principle 5 2.0 Specimen Collection 5

2.1. Special Instructions 5 2.2. Specimen Collection 5 2.3. Sample Quantity 6 2.4. Unacceptable Specimens 6

3.0 Sample Handling 6 3.1. Specimen identity and Integrity 6 3.2. Special Timing Conditions 6 3.3. Transport Conditions 6 3.4. Storage Conditions 7 3.5. Special Equipment 7

4.0 Reagents or Media 7 4.1. Reagents and Sources 7 4.2. Reagent Preparation 8 4.3. Standards Preparation 9 4.4. Preparation of Quality Control (QC) Materials 11

5.0 Test Procedure, Procedure Notes, and Safety Requirements 11 5.1. Test Procedure 11 5.2. Equipment 15 5.3. Supplies 15 5.4. Instrument Configuration 16 5.5. Safety 18

6.0 Calibration and Tuning 20 6.1. Mass Spectrometer Calibration and Tuning 20 6.2. Creation of Standard Curve 22

7.0 Quality Control 24 7.1. Controls to be Used 24 7.2. Preparation and Handling of Control Materials 24 7.3. Frequency with which Control Materials are Run 24 7.4. Establishment of Acceptable Limits for Controls 25 7.5. Corrective Actions when Tolerance Limits are Exceeded 25 7.6. Recording and Storage of QC Data 26

8.0 Calculations 26 8.1. Processing of Data 26

9.0 Reporting Results 27 9.1. Entering Results in LIMS 27 9.2. Reporting Results 27 9.3. Range of Values 27 9.4. Analytical Sensitivity 28 9.5. Accuracy and Precision 28 9.6. Analytical Specificity 28 9.7. Limitations of Method, Interfering Substances and Conditions 28 9.8. Reference Ranges (Normal Values) 28

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9.9. Critical-Call Results (“Panic” Values) 29

10.0 Verification/Establishment Study 29 11.0 Package Inserts Used 29 12.0 MSDS File Location 29 13.0 Proficiency Testing 29

13.1. Enrollment 29 13.2. Frequency 29 13.3. Protocols for Handling PT Samples and Results 29

14.0 Competency Assessment 30 14.1. Description of Method Used to Assess Competency 30 14.2. Frequency 30

15.0 Maintenance 30 15.1. Pipettes 30 15.2. In-House Water Purification System 31 15.3. Mass Spectrometer 31

16.0 Corrective Actions 32 16.1. Pre-Analytic 32 16.2. Analytic 32 16.3. Post-Analytic 32

17.0 References 32

Tables Table 1 – Analytes 5 Table 2 – Chemicals and Sources 7 Table 3 – SC Metabolite Standard Concentrations – Urine 10 Table 4 – SC Metabolite Standard Concentrations – Blood 10 Table 5 – SC Metabolite Standard Concentrations – Tissue 11 Table 6 – LC Flow Rate Program 16 Table 7 – LC-MS/MS Parameters 16 Table 8 – MRM Parameters 17 Table 9 – PPG Data 21 Table 10 – Reportable Range 27

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Synthetic Cannabinoids Metabolites in Urine by LC-MS/MS

1.0 Principle Use of synthetic cannabinoids causes symptoms not typically associated with marijuana and often results in serious injury. Recent research on prevalent synthetic cannabinoids known as AM2201, JWH-018 identified metabolic products in human urine that can be used to detect exposures (Chimalakonda, et. al. & Moran, et.al.) Parent synthetic cannabinoids are not normally excreted in human urine (Chimalakonda, et. al. & Moran, et.al.), but can be found in blood and body tissues. For this reason, the presence of urine metabolites and/or parent compounds in blood and tissues can be used for detection assays. The positive electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS) method described in this standard operating procedure allows for the rapid and accurate quantitation of the parent compounds and metabolites, including the enantiomers of chiral metabolites, used to detect AM2201 and JWH-018 use. The specific analytes are listed in Table 1.

Table 1. Analytes Trade or Other Name Chemical Compound

AM2201 1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone

(R)-(-)-AM2201-(ω-1)-OH (R)-(-)-1-(5-fluoro-4-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-l)methanone

(S)-(+)-AM2201-(ω-1)-OH (S)-(+)-1-(5-fluoro-4-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-l)methanone JWH-018 (1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone

JWH-018-(ω)-OH (1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-yl)-methanone JWH-018-(ω)-COOH 5-(3-(1-naphthoyl)-1H-indol-1-yl)-pentanoic acid

(R)-(-)-JWH-018-(ω-1)-OH (R)-(-)-1-(4-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-yl)-methanone

(S)-(+)-JWH-018-(ω-1)-OH (S)-(+)-1-(4-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-yl)-methanone 2.0 Specimen Collection

2.1 Special instructions No special instructions such as fasting, special diets, etc. are required.

2.2 Specimen collection The laboratory does not collect specimens from patients, but if questions arise, it should be noted that urine specimens should be

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collected from subjects in standard urine collection cups. Although no long term stability studies have been completed for synthetic cannabinoids in urine, other drugs of abuse, such as opiates and stimulants, are known to be extremely stable in urine where little to no degradation of samples occurs at any of the storage temperatures tested, including room-temperature and refrigeration, over the two-week trial period (Frings and Queen). Current clinical laboratories (NMS) report samples are stable for thirty days at room temperature, refrigerated, and frozen. Internal stability studies have also resulted in similar results.

2.3 Sample quantity The optimal amount of specimen is at least 2.0 mL or a minimum volume of 0.5 mL.

2.4 Unacceptable specimens The criteria for an unacceptable specimen include low sample volume (< 0.5 mL), suspected contamination due to improper collection procedures or collection devices, or a leaking or damaged container. A description of reasons for each rejected sample should be recorded in the specimen receiving spreadsheet located on the S:Drive (S:\CT LAB\Sample Receiving\AllCommonSampleLog). In extreme circumstances, the Laboratory Director may override sample rejection.

3.0 Sample Handling 3.1 Specimen identity and integrity

All samples received will be entered into the sample receiving spreadsheet (S:\CT LAB\Sample Receiving\AllCommonSampleLog) located on the S:Drive. The information recorded in the sample receiving log book should at a minimum include: date sample was received in the laboratory, who received the sample, sample identification and description of sample, sample collection date (if known), a description of how the sample was received (i.e. the temperature of the specimen, was the container broken, etc.), storage conditions, and storage location. Additional comments may also be added.

3.2 Special timing conditions

No special timing conditions are required for sample collection for this method.

3.3 Transport conditions Transport conditions have not been established. It is recommended that samples be transported with cold packs, preferably frozen. Special care must be taken in packing to protect the urine cups from

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breakage during shipment.

3.4 Storage conditions All samples should be stored at -80 ± 40°C until analysis. Samples are thawed and aliquoted for analysis; the residual specimen is again stored at -80 ± 40 °C until needed. Specimens may reach and maintain ambient temperature while preparing the sample for analysis. If the measurement is delayed until the next day, specimens should be stored at -80 ± 40 °C. If the analytical system fails, specimens and any partially-prepared/extracted samples can be stored at -80 ± 40 °C until the analytical system is again operational. Repeat measurements of QC samples stored at -80 ± 40 °C indicate that urine samples may be stored for at least 1 year without degradation of the analytes.

3.5 Special equipment No special equipment is necessary for specimen collection or handling. It is generally recommended that urine specimens be collected in standard urine collection cups and/or laboratory containers.

4.0 Reagents or Media

4.1 Reagents and sources Reagents and sources used during the development and validation of this method are listed in Table 2. Specific part numbers and manufacturers are listed only as examples and other sources can be used for purchasing these reagents as long as the quality meets or exceeds the established standards listed in this SOP. All chemicals and solvents are used without further purification. All solid state reagents/standards are acceptable for use for 10 years from the date of open when stored as recommended by the manufacturer unless otherwise indicated through stability studies or by the manufacturer. All liquid state reagents and standards are acceptable for use for 1 year from the date of open when stored as recommended by the manufacturer unless otherwise indicated through stability studies or by the manufacturer.

Table 2. Chemicals and sources Reagent Grade Source * Part Number Acetonitrile Optima Fisher Scientific,

Fairlawn, NJ A996-4

Formic acid Reagent Acros Organic, Pittsburgh, PA

147930010

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Chemistry Laboratory Reagent Water (CLRW)

Organic-pure deionized 18 MΩ water

PureLab Ultra n/a

Beta-glucuronidase Bovine liver, Type B-10, 13,000 units/mg

Sigma-Aldrich, St. Louis, MO

G0501

Sodium acetate ACS Fisher Scientific, Fairlawn, NJ

S609-500

Dimethyl sulfoxide ACS Spectrophotometric

Sigma-Aldrich, St. Louis, MO

D8779

Ammonium Bicarbonate

BioUltra Sigma-Aldrich, St. Louis, MO

09830

Phosphate Buffered Saline (PBS)

n/a Sigma-Aldrich, St. Louis, MO

P4417

* Or equivalent. 4.2 Reagent preparation

At the time of preparation, all reagents should be labeled with name (or initials) of person preparing the solution, expiration date, and contents of solution. Reagents are prepared as needed. Records of all preparations are kept in the K2 Laboratory Notebook. Instructions listed in the following sections (4.2.1 – 4.2.5) are provided as example calculations and care should be taken to ensure final concentrations remain consistent. For example, stock concentrations of purchased materials may vary between vendors, and therefore, dilutions and final protocols have to be adjusted accordingly. 4.2.1 0.1M pH 5 sodium acetate buffer Prepare the buffer by dissolving 6.8 grams of sodium acetate

in 500 mL of CLRW. Check pH of solution using pH meter and adjust pH as necessary using small volumes of 100% acetic acid. This solution can be stored at 4 ± 5 °C indefinitely.

4.2.2 β-glucuronidase in 0.1M pH 5 sodium acetate buffer

Prepare the β-glucuronidase solution by dissolving 5.0 mg β-glucuronidase in 31.65 mL 0.1M pH 5 sodium acetate buffer. This solution can be stored at -80 ± 40°C indefinitely.

4.2.3 Phosphate Buffered Saline (PBS) 1 tablet is dissolved in 200 mL CLRW.

4.2.4 HPLC mobile phases Mobile Phase A: 20mM ammonium bicarbonate. Weigh 1.58

grams of ammonium bicarbonate and mix with 1000 mL

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CLRW. Mobile Phase B: 100% acetonitrile.

4.2.5 Metabolic Standards Metabolic standards are typically obtained from Cayman

Chemical. Equivalent standards may be purchased from another vendor, if necessary. Stock solutions (20 µg/mL) are typically prepared in methanol or dimethyl sulfoxide and stored at -40 ± 10 ºC.

4.2.6 Isotopically labeled internal standard Internal standards (ISTD) are also obtained from Cayman

Chemical. Stock solutions (20 µg/mL) are prepared in either methanol or DMSO and stored at -40 ± 10 ºC.

4.3 Standards preparation At the time of preparation, all standards should be labeled with initials of person preparing the solution, expiration date, and contents of solution. Standards expire as per the manufacturer’s expiration date. All preparations are to be recorded in the K2 Laboratory Notebook. Any time new standard materials are used, ensure that new lots of materials are verified through QC charting. 4.3.1 Intermediate standards

A 1 μg/mL intermediate standard mix solution is prepared if not supplied by the chemical manufacturer. This solution is prepared in either methanol or DMSO and stored at -40 ± 10 ºC.

4.3.2 Working standards

A. Urine (Note: Only prepare if analyzing urine specimens) 1. A 100 ng/mL working standard mix solution is used in

the preparation of the standard curve. It is prepared by adding 100 μL of 1 μg/mL intermediate standard to 900 μL DMSO or methanol. This working standard is serially diluted in base urine known to be free of synthetic cannabinoids to create the standard curve (Table 3). Serial dilution in base urine allows standards to be matrix matched.

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Table 3. SC metabolite standard concentrations – Urine

Concentration (ng/mL)

Previous Standard (uL) Urine (uL)

0.5 125 375

Serial dilutions from 100 ng/mL

working standard

2 200 300 5 250 250 10 200 300 25 250 250 50 250 250

B. Blood (Note: Only prepare if analyzing blood specimens)

1. A 10 ng/mL, 100 ng/mL, and 1000 ng/mL working standard mix solution are used in the preparation of the standard curve. The1000 ng/mL solution is taken from the intermediate standard, and the others are prepared by a serial dilution of this solution. These working standards are diluted in blank blood known to be free of synthetic cannabinoids, and the standard curve is created as described in Table 4. Dilutions in blood allow standards to be matrix-matched.

Table 4: SC metabolite standard concentrations – Blood Concentration

(ng/mL) Standard (uL) Blood (uL)

0.2 2 100 Use 10 ng/mL working standard 0.5 5 100 2 20 100

5 5 100 Use 100 ng/mL working standard 10 10 100

25 25 100 50 5 100 Use 1000 ng/mL

working standard 100 10 100 C. Tissues (Note: Only prepare if analyzing tissue specimens)

1. A 100 ng/g working standard is made by adding 500 μL of 1 μg/mL intermediate standard to 4500 μL of blank tissue homogenate. The blank tissue homogenate is prepared by homogenizing tissue known to be free of synthetic cannabinoids in 5X volume of PBS (i.e., 1 g tissue + 5 mL PBS). This working standard is serially diluted in blank tissue homogenate to create the standard curve (Table 5). Serial dilution in tissue homogenate allows standards to be matrix matched.

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Table 5: SC metabolite standard concentrations – Tissue

Concentration (ng/mL) Standard (uL)

Tissue Homogenate

(uL)

0.2 400 600

Serial dilutions from 100 ng/g

working standard

0.5 250 750 2 400 600 5 500 500 10 400 600 25 500 500 50 500 500

4.4 Preparation of quality control (QC) materials

All QC materials are prepared from certified, traceable standards that are obtained from Cayman Chemical, or equivalent. Working QC standards are made in the same manner as the standards used to build standard curves (See tables 3-5). If possible, the QC material should be prepared from a second source (either a different manufacturer or a different lot number if obtained from the same manufacturer) but in some cases only one source may be available. Regardless, QC material is always prepared separately from standards. Each of these QCs should be evaluated with each run.

4.4.1 Urine QCs are titered to deliver 0, 2, and 50 ng metabolite per mL urine for the Quality Control Blank (QCB), Quality Control Low (QCL), and Quality Control High (QCH), respectively.

4.4.2 Blood QCs are titered to deliver 0, 2, and 50 ng metabolite per mL urine for the Quality Control Blank (QCB), Quality Control Low (QCL), and Quality Control High (QCH), respectively.

4.4.3 Tissue QCs are titered to deliver 0, 2, and 50 ng metabolite per g tissue for the Quality Control Blank (QCB), Quality Control Low (QCL), and Quality Control High (QCH), respectively.

5.0 Test Procedure, Procedure Notes, and Safety Requirements

An analytical run consists of a blank, standards, quality control samples, and unknown urine samples. NOTE: Time, temperature, and RPM are non-critical parameters used in this test procedure and do not need to be verified. 5.1 Test procedure

5.1.1 Sample preparation

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A. Urine Samples (Note: Only perform if analyzing urine

specimens) 1. Allow unknown urine specimens, matrix-matched

standards, and QC samples to reach ambient temperature.

2. Vortex the urine samples, standards, and QCs and

pipette 80 μL into the bottom of each appropriately-labeled 1.5 mL Eppendorf tube. Add 40 μL of Internal Standard and vortex until well-mixed.

3. To each sample, add 320 μL β-glucuronidase in 0.1M

pH 5 sodium acetate buffer. Vortex all samples until well mixed.

4. Place tubes in a shaking incubator at 37°C, 700 RPM

for approximately 1 hour. Samples should be immediately analyzed but can be stored at -80 ± 40 °C for up to 30 days prior to analysis.

B. Blood Samples (Note: Only perform if analyzing blood

specimens) 1. Allow unknown blood specimens, matrix-matched

standards, and QC samples to reach ambient temperature.

2. Vortex the blood samples and pipette 100 µL into the bottom of each appropriately-labeled 1.5 mL Eppendorf tube. Add 10 µL of Internal Standard and vortex until well-mixed.

3. To each tube, add 400 µL acetonitrile and vortex for approx. 30 seconds.

4. Place the tubes in -40 ± 5°C for 30 minutes.

5. Vortex until well mixed and centrifuge at max RPM for 10 minutes.

6. Transfer supernatant into an appropriately-labeled glass test tube and evaporate under a stream of nitrogen at 45± 5°C until completely dry.

7. Reconstitute in 100 µL DMSO. Samples should be immediately analyzed but can be stored at -80 ± 40

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°C for up to 30 days prior to analysis.

B. Tissue Samples (Note: Only perform if analyzing tissue specimens)

1. Allow unknown tissue specimens, matrix-matched standards, and QC samples to reach ambient temperature.

2. Homogenize unknown tissue samples in 5X volume of PBS buffer (i.e., 1 g tissue + 5 mL PBS).

3. Vortex the tissue samples, standards, and QCs, and pipette 200 µL into the bottom of each appropriately-labeled 15 mL conical. Add 10 µL of Internal Standard and vortex until well-mixed.

4. To each tube, add 800 µL acetonitrile and vortex for approx. 30 seconds.

5. Place the tubes in -40 ± 5°C for 30 minutes.

6. Vortex until well mixed and centrifuge at max RPM for 10 minutes.

7. Transfer supernatant into an appropriately-labeled glass test tube and evaporate under a stream of nitrogen at 45± 5°C until completely dry.

8. Reconstitute in 100 µL DMSO. Samples should be immediately analyzed but can be stored at -80 ± 40 °C for up to 30 days prior to analysis.

5.1.3 Sample analysis 1. Preliminary system setup and performance check

a. Check the reservoirs on the HPLC to ensure sufficient mobile phase is present.

b. Check the column oven to ensure the correct analytical

column (Lux 3u Cellulose-3 – Phenomemex Part # 00F-4492-B0, or equivalent) and guard column (SecurityGuard Cartridges Lux Cellulose-3 – Phenomemex Part # AJ0-8621, or equivalent) are installed correctly.

c. Connect the HPLC to the mass spectrometer.

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d. Check the needle rinse solvent level to ensure there is

plenty of methanol to perform the run. e. Select the “SC Chiral” project folder from the drop-

down menu. f. It is recommended to inject the solvent blank (100%

DMSO) first to ensure the baseline is clean and then inject the QC blank to ensure sufficient counts of the internal standards are present. If a check sample (previously used standard or sample) is available, it can be injected to test the system performance.

5.1.3 Final set-up and operation

1. Check that the sample vials are in the autosampler tray. Be sure that the autosampler chamber is set at room temperature. NOTE: Before loading vials into the autosampler be sure to evacuate all air bubbles from the bottom of the vial insert.

2. Double click on Build Acquisition Batch in the Analyst

software. Click on Add Set, then Add Samples to enter the appropriate number of samples. Enter the names of the standards (Urine Std. 0.5 ng/mL – Urine Std. 50 ng/mL), QCs (QCB, QCL, QCH), and unknown samples in the Sample Name column. Unknown samples will be labeled with a unique identifier (i.e., sample number). If using LC vials, select 54-vial plate; if using 96-well plate, select the plate type. Select the acquisition method (Chiral_Analysis_A1B1 OR Chiral_Analysis_A2B2). A1B1 and A2B2 refer to the nomenclature used to select appropriate mobile phase reservoirs.

3. A solvent blank is generally run before the standard curve,

then the standard curve is run, then a solvent blank, followed by the QCs. If carryover is suspected, the analyst may choose to run a solvent blank between samples. Bracket at least every 20 samples with a quality control sample and a blank. Incorporation of these additional quality control samples assists in evaluating performance and carryover throughout the entire run.

4. Verify that the positions of samples entered in the batch

correspond to the samples in the autosampler. 5. Select the Submit tab then the submit button.

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6. Select the View Queue icon, and then select the Run

Sample icon.

5.1.4 System shutdown The instrument will automatically go into Standby mode shortly after the analysis has finished.

5.2 Equipment 5.2.1 LC-MS/MS

The LC-MS/MS analyses are performed on an Agilent 1200 quaternary liquid chromatography system (Santa Clara, CA) coupled to an AB Sciex API-4000 Q-Trap tandem mass spectrometer (AB Sciex, Carlsbad, CA).

5.2.2 Wellplate autosampler The autosampler is configured to sample from 54-position vial trays. An injector needle wash is performed by using a custom injector program. The injection program picks up 10 μL of sample, washes the injector in the flush port for 5 sec, and injects the sample. The wash solvent is HPLC grade methanol.

5.2.3 HPLC column oven The column oven is held at ambient temperature.

5.2.4 HPLC degasser The degasser is set on.

5.3 Supplies Supplies should meet or exceed the listed requirements if procured from these or other sources. These are only listed as examples to aid in reordering. 1.5 mL Eppendorf tubes CLRW (>18.2 Mega-ohm*cm, ultrapure) Kim-WipeTM tissues Nitrile gloves Vortexer (VWR, West Chester, PA) Homogenizer Thermomixer (Eppendorf, Hauppauge, NY) Pipettes, 1-mL and 100-μL (Rainin Instrument, Woburn, MA) Inline Filter, 0.5-μm, 0.5-μL PEEK (Upchurch Scientific, Inc. Oak

Harbor, WA) PEEK tubing, 1/16” OD, 0.005” ID (Upchurch Scientific, Inc. Oak

Harbor, WA)

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PEEK Fittings, ¼-28, 1/16” (Upchurch Scientific, Inc. Oak Harbor,

WA) Assorted glassware

5.4 Instrument configuration 5.4.1 Binary pump configuration

The binary pump is configured to run a gradient, see Table 6.

Table 6. LC flow rate program Time (min.) % A % B Flow Rate (μL/min)

0 60 40 500 10 5 95 500 12 5 95 500 15 60 40 500 16 60 40 500

5.4.2 LC-MS/MS configuration

The LC-MS/MS configuration is described in Table 7 below. Mass spectrometer settings may vary; typical settings are shown below.

Table 7. LC-MS/MS Configuration

Parameter Setting

Column Type Phenomemex Lux 3u Cellulose-3 – Part # 00F-4492-B0, or equivalent

Guard Column Type Phenomemex SecurityGuard Cartridges Lux Cellulose-3 – Part # AJ0-8621, or equivalent

Mobile Phase Mobile Phase A: 20mM ammonium bicarbonate Mobile Phase B: 100% acetonitrile See table 4 for LC gradient

Mass spectrometer mode Positive electrospray ionization, multiple reaction monitoring (MRM)

Curtain Gas (CUR) 35.0 cm/S [Nitrogen]

Nebulizer Gas (GS1) 55 cm/S [Nitrogen]

Turbo Gas (GS2) 55 cm/S [Nitrogen]

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GS2 Temperature (TEM) 600 ºC

Interface Heater (ihe) ON

Collision Gas (CAD) “high” [Nitrogen]

Ionspray Potential (IS) 2500 V

Entrance Potential (EP) 10 V

Column Oven Right Temperature 40 ºC

Column Oven Left Temperature 40 ºC 5.4.3 MRM configuration

A multiple reaction monitoring (MRM) experiment is initiated by the HPLC controller at the time of injection. The MRM transitions listed in Table 8 (along with compound-dependent parameters). Mass spectrometer settings may vary; typical settings are shown in Table 8. An information dependent acquisition-enhanced product ion (IDA-EPI) experiment is also included to confirm the presence of each metabolite. The specific conditions of this experiment are included in Table 8.

Table 8. MRM parameters

Analyte Q1

(m/z) Q3

(m/z)

Collision Energy

(V)

Entrance Potential

(V)

Declustering Potential

(V)

Collision Cell Exit Potential

(V)

SRM

AM2201 360 155* 71 10 106 22

360 127# 35 10 106 6

(R)-(-)-AM2201-(ω-1)-OH

376 155* 37 10 81 10

376 127# 77 10 81 6

(S)-(+)-AM2201-(ω-1)-OH

376 155* 37 10 81 10

376 127# 77 10 81 6

AM2201-(ω-1)-OH-d5 381 155* 37 10 106 8

381 127# 87 10 106 18

JWH-018 342 155* 37 10 86 12

342 127# 67 10 86 8

JWH-018-(ω)-OH 358 155* 37 10 86 12

358 127# 67 10 86 8

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JWH-018-(ω)-OH-d5 363 155* 33 10 141 10

363 127# 77 10 141 8

JWH-018-(ω)-COOH 372 155* 37 10 86 12

372 127# 67 10 86 8

JWH-018-COOH-d4 376 155* 37 10 86 12

376 127# 67 10 86 8

(R)-(-)-JWH-018-(ω-1)-OH

358 155* 37 10 86 12

358 127# 67 10 86 8

(S)-(+)-JWH-018-(ω-1)-OH

358 155* 37 10 86 12

358 127# 67 10 86 8

JWH-018-(ω-1)-OH-d5 363 155* 33 10 66 10

363 127# 81 10 66 18

JWH-018 Oxidation + Glucuronidation 534 358

^ 30 10 30 15

JWH-018 Carboxylation + Glucuronidation

548 372^ 30 10 30 15

IDA-EPI 1-14 [MH]

+ 80-600 40 10 40 N/A

* Quantitation Ion # Confirmation ion ^ No quantitation performed on these ions. These are strictly monitored to assess the efficiacy of β-Glucuronidase reactions.

5.4.4 LC-MS/MS instrument control program An instrument control program is created using the current software version of Analyst based upon the parameters described above.

5.5 Safety All general safety precautions outlined in the Arkansas Department of Health Public Safety Manual should be followed for this method. 5.5.1 Reagent toxicity or carcinogenicity

Little information is known about the toxicological nature of synthetic cannabinoids and their metabolites. The synthetic cannabinoid metabolites are not known to be carcinogenic.

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Care should be taken to avoid inhalation or dermal exposure to acetonitrile and DMSO.

5.5.2 Radioactive hazards None.

5.5.3 Microbiological hazards Follow universal and general laboratory precautions. Because of the possibility of being exposed to various microbiological hazards, appropriate measures should be taken to avoid any direct contact with the urine specimens. Gloves, lab coats, and safety glasses must be worn while handling all human urine products. A Hepatitis B vaccination series is recommended for health care and laboratory workers who are exposed to human fluids and tissues.

5.5.4 Mechanical hazards There are only minimal mechanical hazards when performing this procedure using standard safety practices. Laboratorians should read and follow the manufacturer’s information regarding safe operation of the equipment. Avoid direct contact with the mechanical or electronic components of the automated sample preparation instrument, liquid chromatograph and mass spectrometer, unless all power to the instrument is off. Be aware that the mass spectrometer employs a high voltage, low amperage ionization source. Generally, mechanical and electronic maintenance and repair should be performed only by qualified technicians.

5.5.5 Protective equipment Standard safety precautions should be utilized when performing this procedure. These precautions may include use of lab coat, safety glasses, face mask, durable gloves, and a chemical or biological fume hood. Refer to the Chemical Hygiene Plan for details related to specific activities, reagents, or agents.

5.5.6 Personal hygiene

Follow universal and general laboratory precautions. Care should be taken in handling any biological specimen. Routine use of gloves and proper hand washing should be practiced. Refer to the Chemical Hygiene Plan for details related to specific activities, reagents, or agents.

5.5.7 Disposal of wastes

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Waste materials must be disposed of in compliance with laboratory, federal, state, and local regulations. All disposable items that come in direct contact with the urine specimens are to be placed in a biohazard autoclave bag that should be kept in appropriate containers until they are removed for disposal. Wipe down all surfaces and non-disposable equipment with a 90% ethanol solution (or equivalent) and allow to sit for 10 min when work is finished.

6.0 Calibration and Tuning 6.1 Mass spectrometer calibration and tuning

Mass spectrometer calibration and tuning should be performed at least annually as part of the preventative maintenance (PM) plan per the annual service agreement. Tuning may be performed more often when measured values for QCL and QCH fall outside of the accepted laboratory values. Tuning can be performed manually (described in Section 6.1.5) or automatically. Manual tuning should only be performed by laboratory personnel who are trained in operation of the API 4000 Q-Trap. Calibration of the mass spectrometer is to be performed by a service engineer, but can be performed internally by trained individuals. 6.1.1 Preparation of the tuning standard

The mass spectrometer is tuned using the PPG 4000 standard solution (Applied Biosystems).

6.1.2 Acceptable tolerances The mass value should be accurate to 0.1 Dalton, and peak widths should be 0.7-0.8.

6.1.3 What to do when tolerances are not met The instrument should be re-tuned when the mass values or peak widths fall outside the accepted range. If necessary, call the instrument manufacturer for technical assistance and/or instrument repair as covered by the service agreement.

6.1.4 Tuning procedure 1. Place LC in standby mode.

2. Activate the appropriate hardware configuration –

MassSpecOnly

3. Activate the tune menu to initialize the instrument by selecting Tune on the left side of the screen. Select Manual Tune.

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4. Open “API Instrument” Project from drop-down list

5. Open “Q1 Pos PPG”

6. Load the 1 mL syringe with the 4000 Pos PPG (2x10-6 M)

solution, slide the syringe into the tubing union with sleeve, and place in the syringe pump. Make sure pumping mechanism is set firmly against syringe.

7. Attach the syringe to the ESI source with PEEK tubing.

8. Set the infusion rate to10 μl/min and begin infusion.

9. Wait a few minutes, allowing the PPG solution to pump to the MS.

10. Assure that the correct information is listed in the table: Table 7: PPG Data

Center (Da) Width (Da) Time (sec) 59.050 6 0.6100

175.133 6 0.6100 616.464 6 0.6100 906.673 6 0.6100 1254.925 6 0.6100 1545.134 6 0.6100 2010.469 6 0.6100 2242.637 6 0.6100

11. Right click on Q1 spectrum window. Select Open file.

12. Right click on any graph and select List Data.

13. Click on Calibration List Peak tab.

14. To ensure correct reference, right click on table and select

PPGs Pos. Calibration and then select Use as reference.

15. Adjust Peak Width a. If peak widths are outside the acceptable 0.7-0.8

Dalton range, close current window to return to Manual Tune window.

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b. Select Resolution tab and click the Advanced button

to open the Resolution Table Editor.

c. Change offset to improve peak width. (Smaller offset results in wider peak.) Click off of active cell then click Apply.

d. Acquire new data by clicking the Start button. Repeat steps 11-15 until desired peak width is met.

e. Once peak width falls within normal range, the instrument may be recalibrated if necessary. Select any graph following the Open file command in step 11.

16. Adjust Mass Shift a. If mass shift is > 0.2, the instrument must be

recalibrated.

b. Click on any graph and press the Calibrate from spectrum button on the toolbar above the open window.

c. Choose PPGs Pos., Search range 4, Threshold 200 cps. Select OK.

d. This brings up the calibration graph. If the data points fall within the solid line and the dashed line, press the Replace button at the top left of the window.

e. Close the calibration graph window. This will bring up the slope variation data. The slope variations must fall between 0.998 and 1.002 for the instrument to maintain proper sensitivity. If these are off, adjust peak width until all fall within normal range.

17. Elect to save the new settings when prompted.

18. Discard the excess tuning solution, rinse the syringe four times with methanol, and rinse the tubing (and ESI sprayer) with methanol from the syringe.

19. Reconnect HPLC to ESI source.

20. Reset Hardware configuration back to original setting.

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6.2 Creation of standard curve

Standard responses are achieved using standard solutions prepared in Section 4.3.2. A linear standard curve is used for the seven metabolites and generated by normalizing instrument response of each analyte to the response of the internal standard.

6.2.1 Data collection A standard curve is established for each run by plotting the normalized standard response to the theoretical concentration.

6.2.2 Calculation of curve statistics The slope, intercept, and R-value for the standard curve are generated using linear regression with 1/x weighting. The slope and intercept of the standard curve are determined by linear least squares fit using the Analyst software provided with the instrument.

6.2.3 Evaluation of curve statistics The R-squared value for the curve must be ≥ 0.99 for a run to be considered acceptable. Linearity of the standard curve should extend over the entire standard range. The intercept is calculated from the least squares fit of the data. One standard may be removed if there is an obvious error; however, the same standard should not fail in consecutive analytical runs. If consecutive runs fail, investigations may need to be conducted to identify the source of error. The lowest and highest standards can not be removed from any analytical run because these are critical for reporting results. If errors occur with those standards then new standards and matrix-matched QCs need to be prepared and evaluated along with patient specimens.

6.2.4 Standard curve verification As part of each analytical run, a curve is constructed from the six standards. Verification is conducted by quantifying Quality Control samples of known value against the standard curve and statistically comparing the calculated results using quality control charting and Westgard rules of statistical evaluations.

6.2.5 Usage of curve If an analyte concentration is observed that is higher than the highest standard concentration, then report as greater than highest calibrator. If an analyte concentration is lower than the lowest standard concentration, then report as less than the minimum reporting limit. In every case, metabolites must be

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confirmed before reporting by comparing response ratios of quantifying and confirmation ions

7.0 Quality Control Quality assessment procedures follow standard practices. As noted above, daily experimental checks are made on the stability of the analytical system. In addition, blanks and standards, as well as QC materials are added to each day's run sequence. A standard curve is developed for the batch using a complete set of standards and evaluated as described in section 6.2. The results from the analysis of the QC material(s) obtained using this standard curve is compared with acceptance criteria to assure the proper operation of the analysis. 7.1 Controls to be used

A QC blank, 2 ng/mL, and 50 ng/mL control are to be incorporated in to each analytical batch. These controls are to be run prior to analyzing patient samples, at least every 20 samples throughout the run, and at the end of the run to bracket any unknown/patient samples. Acceptability of results for the entire analytical run is dependent upon the agreement of the results from the QC materials within established ranges as determined through QC charting. The following criteria are the minimum requirements for evaluation of the QC results: A QC result is considered “out of control” if the mean QC concentration is outside of the three-σ (standard deviation, SD) confidence interval, or if two consecutive mean QC concentrations are outside the same 2-σ confidence interval.

If a QC result is “out-of-control,” the cause of the failure must be determined and corrected. Sometimes the cause of failure may be related to statistical probability and no additional measures may need to be taken in the laboratory. Additional QC may be added to analytical batches to fully assess accuracy and precision and may be used as quality control data. In these cases, data may be reported. The analyst, supervisor, and section director will review each batch with a QC error and make determinations on the data as appropriate.

7.2 Preparation and handling of control materials Quality Control (QC) samples are prepared in urine as described in Section 4.4.

7.3 Frequency with which control materials are run A QC blank, 2 ng/mL, and 50 ng/mL control are to be incorporated in to each analytical batch. These controls are to be run before prior to analyzing patient samples, at least every 20 samples throughout the

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run and at the end of the run to bracket any unknown/patient samples.

7.4 Establishment of acceptance limits for controls

Quality Control limits will be established during the method validation and evaluated annually during method validation studies. As indicated above, QC results are tracked through continuous control charting and statistical evaluations.

7.5 Corrective actions when tolerance limits are exceeded

If standard or QC systems fail (e.g. not being able to report results), all operations must be suspended until the source or cause of failure is identified and corrected. Analytical results are typically not reported for runs not in statistical control unless otherwise instructed by the Laboratory Director or Supervisor. Before beginning another analytical run, an appropriate corrective action will be initiated. Once standard verification and/or quality control have been reestablished, samples must be re-run. All Corrective Action Reports (CARs) are tracked through the laboratory QA/QC Office. http://phl_server7/ca/castart.aspx 7.5.1 Low area counts in internal reference

Signal/noise ratio for all analytes should exceed 10. If S/N falls below 10, this indicates a problem with instrument sensitivity. The following steps should be taken and the instrument sensitivity rechecked after each is performed. 1. Clean mass spectrometer curtain gas plate and orifice. 2. Replace the electrospray ionization capillary.

3. Prepare fresh HPLC mobile phase. 4. If sensitivity is lowered due to band broadening, inspect all

HPLC connections and consider changing the column.

Once sensitivity has been reestablished further steps are not necessary.

7.5.2 Linearity criterion not met If the linearity criterion of greater than or equal to 0.99 is not met, check that there is not one standard that is grossly in error. This can be caused by improper standard preparation or instrument malfunction. If no standard is found in error, check for nonlinearity due to detector saturation In this event, instrument re-tuning may be required.

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7.5.3 High analyte in one standard If an inordinately large amount of analyte is measured in one of the standards or QC materials, but this is not seen in the remainder of the samples, this indicates a contamination of this particular sample. The source of this incident should be investigated to prevent repeat occurrences, but no further action is required.

7.5.4 High analyte in all samples If an inordinately large amount of analyte is present in all measurements for a particular day, it is likely that one or more of the spiking solutions are contaminated. If necessary, prepare new solutions.

7.5.5 Measurement outside confidence limits If the concentration of the QC sample falls outside the ±3σ confidence limits, check that the internal standard was added to that sample. Also confirm that the integration was performed correctly. Finally, make sure that the correct internal standard level is designated in the quantitation software, and that proper dilution factors are considered.

7.6 Recording and storage of QC data Data analysis is performed using Analyst software as described in Section 8.0. Data are stored in both hard copy and electronic formats.

8.0 Calculations

8.1 Processing of data All raw data files are quantified using the quantitation software package in Analyst. The data processing results are saved in a quantitation file (.rdb). This file contains a graphic depiction of the retention time window and the area integrated for the analyte and the internal standard for each sample. The file shows the standard curve used and its R value. The integrated peak areas for each analyte, retention times, and concentrations are saved in a text file. The text files are saved with the same name as used in the .rdb file. 8.1.1 Analyzing and storing the LC-MS/MS data

1. In Analyst, choose Quantitation Wizard and the data set and add the desired samples. Choose the correct quantitation method (Chiral_Quant.qmf) and select Finish. All of the samples will be processed and ready for review. The standard curve line should not be forced through the origin but the curve should be 1/X weighted.

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2. The analyst should review the S/N ratios (>10), EPI data,

and retention times of peaks that are very low in concentration to verify that the correct peak has been chosen for quantitation and that the peak is actually a quantifiable peak.

3. Choose the Peak Review window. Check the integration of each. Manual integration may be necessary if the software does not choose the correct peak for integration or if a portion of the peak is omitted from the integrated area. Save the .rdb table with the current date.

9.0 Reporting Results

9.1 Entering results in LIMS Not valid for this method.

9.2 Reporting results Results should be reported to meet client needs.

9.3 Range of values The method detection limits and minimum reporting limits are determined during the validation studies and LOD is calculated as 3 times the standard deviation of QCL. In all cases, signal to noise should be at least 10 for both quantifying and confirmation ions listed in Table 8. Urine metabolite values are reportable in the range between the lowest quantified standard and the highest quantified standard. The lower reportable limit is set at the concentration of the lowest standard in the standard curve. The upper reportable limit is the concentration of the highest standard.

Table 9. Reportable range

Compound Method LOD*

Lower reportable limit*

Upper reportable limit*

AM2201-(ω-1)-OH

JWH-018-(ω)-OH

JWH-018-(ω)-COOH

JWH-018-(ω-1)-OH

JWH-073-(ω)-OH

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JWH-073-(ω)-COOH

JWH-073-(ω-1)-OH

*Will be established during method validation studies. 9.4 Analytical Sensitivity

The method detection limit is defined in Section 9.3. However, the LOD should be confirmed by evaluating quantifying and confirmation ion responses. Signal-to-noise should be greater than 10 for both ions and adjusted accordingly during the method validation study

9.5 Accuracy & Precision Accuracy and precision data will be located in the validation plan results. The data will be updated annually through required continuing validation as monitored by QC charting. In general, accuracy should be maintained within ± 30% of target values and %CV should be less than 30%.

9.6 Analytical Specificity

The LC-MS/MS analysis provides excellent analytical specificity. The analyte peaks are located in well defined regions of the chromatogram with no visible interferences and very low background. EPI experiments also provide added specificity and help ensure interferences are not present.

9.7 Limitations of Method; Interfering Substances and Conditions Care is required in order to prevent contamination of QC materials, standards, and samples. This method is an isotope dilution mass spectrometry method, widely regarded as the definitive method for the measurement of organic toxicants in human body fluids. Using unit resolution tandem mass spectrometry eliminates most analytical interferences. Due to the nature of the matrix analyzed in this procedure, occasional interferences from unknown substances might be encountered. Interference with the reference standards results in rejection of that analysis. If repeating the analysis does not remove the interference with the reference standard, the results for that analyte are not reportable.

9.8 Reference Ranges (Normal Values) There is no natural environmental exposure that is known to produce the synthetic cannabinoid metabolites in urine, therefore the reference range is expected to approach zero for non-exposed persons.

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9.9 Critical-Call Results (“Panic” Values)

Early manifestations of synthetic cannabinoid toxicity include extreme agitation, tachycardia, hallucinations, and syncope. The fatal threshold for synthetic cannabinoid exposure is difficult to assess due to factors such as route of exposure, variability of elimination half-lives, storage conditions prior to analysis, and effects of antidotes. Any positive value should be considered critical and reported using normal laboratory reporting procedures.

10.0 Verification/Establishment Study The method is re-verified annually through detection limit studies. Results are stored both electronically and in hard copy formats located in the laboratory.

11.0 Package Inserts Used

Not applicable for this method.

12.0 MSDS File Location All MSDS’s are housed within the Laboratory Safety Section and can be obtained from the Laboratory Safety Officer. There is also an MSDS folder in the CT Laboratory and/or located on the S:Drive (S:\CT LAB\MSDS)

13.0 Proficiency Testing (PT)

13.1 Enrollment At this point, proficiency testing is not provided for synthetic cannabinoids. Proficiency testing will be conducted internally at least on a semi-annual basis. In the event an acceptable external proficiency provider becomes available, the laboratory will participate in these exercises.

13.2 Frequency Proficiency testing is performed at least twice a year.

13.3 Protocols for handling PT samples and results 13.3.1 Sample handling

Samples are handled according to established laboratory protocol. Proficiency testing samples are received by the analyst and taken to the laboratory. As soon as samples are received by the laboratory, they are stored as described in section 3.3. Analyst is to record specimen collection information in the All Common Log on the S:Drive (All Common Sample Log) and the QA Office is provided with the name of the proficiency test, the date of arrival, and the due date so as to enter this information into the PT tracking log http://phl_server7/pt/.

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13.3.2 Reporting Results to the QA Office

After analysis of PT samples has been completed, the PT results will be reviewed by the analyst and Section Supervisor or designee. Laboratory personnel will update the PT tracking once results have been submitted. After graded results have been received by the submitter, the QA Office is provided with a copy of the results in order to update the PT tracking log.

13.3.3 Reporting PT results to the CDC Not applicable for this method.

14.0 Competency Assessment

14.1 Description of method used to assess competency Competency is assessed by direct observation of the analyst performing the method, assessing problem solving skills, and reviewing data. PHL-12-100 is completed during direct observation and is maintained by the QA office.

14.2 Frequency Competency is assessed for new employees once they demonstrate their ability to perform the analytical method. It is re-assessed for new employees at 6 months and then a year. After an employee’s initial year competency, competency is assessed every year thereafter.

15.0 Maintenance Any instrument requiring maintenance should be logged into the Instrument Maintenance Log Excel file S:\CT LAB\Instrument Logs\Instrument Maintenance Log.xls. Daily performance evaluations are typically done during these events to ensure instruments are stable. Preventative maintenance is performed annually by a trained service engineer on instruments used for this method. In most cases, this service is provided under a manufacturer service agreement. 15.1 Pipettes

15.1.1 Calibration All pipettes in use in the laboratory must be calibrated prior to initial use (by manufacturer) and every year thereafter (in-house or by manufacturer).

Pipettes may be calibrated by the manufacturer and the Certificate of Analysis kept in a file for that pipette. Tolerance limits are provided in manufacturers’ calibration reports. No

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further calibration is necessary if pipette is used within 1 year of certificate date.

15.1.2 Corrective Action If delivery of pipetter does not fall within the specified tolerance limits, the pipetter must be adjusted (in-house or by manufacturer), used for less-critical delivery, or discarded. Following pipetter adjustment, a minimum of 10 weighings must be done to evaluate accuracy.

15.2 In-house Water Purification System The laboratory has a Pure Lab Type I Water System (Elga PureLab Ultra). This is also referred to the “chemistry laboratory reagent water,” CLRW in this SOP. Sanitization should be performed monthly according to the protocol outlined in the Purification System Manual. Filters will be changed every 6 months or as needed to meet specifications by trained personnel. The water system is inoperable if the daily measured and recorded resistance is not 18.2 MΩ·cm. A corrective action must be initiated if the system is not operating properly. Water samples do not have to be submitted to Clinical Microbiology for total heterotrophic plate count testing, as the presence of microorganisms do not interfere with this analysis.

15.3 Mass Spectrometer 15.3.1. Cleaning

The sample cone and TurboIon Spray probe should be checked for cleanliness before each run. If dirty, clean the sample interface and Turbolon spray probe as outlined below. The orifice, skimmer, and Q0 should be cleaned as needed when instrument sensitivity falls below manufacturer specifications. Powder free gloves should be worn when handling all parts, and cleaning should be performed with lint free wipes (i.e. KimWipes).

15.3.2. Sample Cone

The sample cone is removed from the instrument by grasping firmly and pulling straight away from the instrument. It is then rinsed sequentially with methanol and CLRW, wiping with a lint free wipe between each rinse. When handling the sample cone, be extremely careful to wipe away from the hole in the center of the cone.

15.3.3. TurboIon Spray Probe

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The TurboIon Spray probe is removed from the source and cleaned by wiping the outside sequentially with CLRW and methanol-soaked lint-free wipes. The electrode tube is removed from the probe by removing the PEEK tubing, placing a blanking plug or nut into the Teflon block, and pulling gently. Care must be taken at this step to not bend the electrode or to lose the spring from inside the probe. The electrode tube is then cleaned sequentially with CLRW and methanol-soaked lint-free wipes. If any discoloration or pitting is noted on the electrode tube, it should be replaced.

15.3.4. Orifice, Skimmer, and Q0

These parts are cleaned only if instrument sensitivity has dropped to below manufacturer specifications (found in the instrument software guide). The instrument must be vented to clean these parts. Follow the procedure outlined in the instrument hardware guide.

16.0 Corrective Actions Corrective actions will be written for any of the following errors made in the

pre-analytic, analytic, or post-analytic stage of testing. Corrective actions are initiated and tracked at the following site: http://phl_server7/ca/castart.aspx 16.1 Pre-analytic

1. Failure to reject specimens when pre-analytic requirements have not been met

2. Improper storage of samples 3. Failure to promptly notify submitters and laboratory section

director when a specimen is rejected 4. Failure to comply with pre-analytic SOP requirements.

16.2 Analytic 1. Any QC or failure of PT due to testing errors 2. Failure to comply with analytic SOP requirements

16.3 Post-analytic 1. Reporting of incorrect results 2. Resampling requests caused by laboratory errors 3. Failure to comply with post-analytic SOP requirements

17.0 References

Chimalakonda, K., et. al. 2011. “Solid-phase extraction and quantitative measurement of omega and omega-1 metabolites of JWH-018 and JWH-073 in human urine.” Analytical Chemistry 83 (16), pp 6381-6388.

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Moran, C., et. al. 2011. “Quantitative Measurement of JWH-018 and JWH-

073 Metabolites Excreted in Human Urine” Analytical Chemistry 83 (11), pp 4428-4236.

Frings, C.S. and Queen, C.A. 1972 “Stability of certain drugs of abuse in

urine specimens” Clinical Chemistry 18 (11), pp1442.