INSERT LABORATORY NAME HERE

Standard Operating Procedure

for

Low-Level Quantitative Analysis of THC and its Metabolites in Blood

by LC-MS/MS

Date: 04/20/16Page 2 of 23

Table of Contents

1.0 Principle 4Table 1. Analytes.......................................................................................................4

2.0 Specimen Collection 42.1 Special instructions 42.2 Specimen collection 42.3 Sample quantity 42.4 Unacceptable specimens 4

3.0 Sample handling 53.1 Specimen Identity and Integrity 53.2 Special timing conditions 53.3 Transport conditions 53.4 Storage conditions 53.5 Special equipment 5

4.0 Reagents or Media 54.1 Reagents and sources 5

Table 2. Chemicals and sources............................................................................64.2 Reagent Preparation 64.3 Preparation of Standards 74.4 Preparation of Quality Control (QC) Materials 7

5.0 Test Procedure, Procedure Notes, and Safety Requirements 75.1 Test Procedure 75.2 Equipment 95.4 Instrument Configuration 10

Table 3. Binary Pump Configuration....................................................................10Table 4. LC-MS/MS Configuration.......................................................................11Table 5. Analyte List with Transitions..................................................................11

5.5 Safety 126.0 Calibration 13

6.1 Mass Spectrometer Calibration 136.2 Creation of standard curve 14

7.0 Quality Control 157.1 Controls to be Used 157.2 Preparation and Handling of Control Materials 16

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7.3 Frequency with which Control Materials are Run 167.4 Establishment of Acceptance Limits for Controls 167.5 Corrective Actions when Tolerance Limits are Exceeded 167.6 Recording and Storage of QC Data 17

8.0 Calculations 188.1 Processing of data 18

9.0 Reporting Results 189.1 Reporting results to Clients 189.2 Range of values 18

Table 6. Linear Ranges..........................................................................................189.3 Analytical Sensitivity 189.4 Accuracy & Precision 199.5 Limitations of Method; Interfering Substances and Conditions 199.6 Reference Ranges (Normal Values) 199.8 Critical-Call Results (“Panic” Values) 19

10.0 Verification/Establishment Studies 1911.0 Package Inserts Used 1912.0 SDS File Location 1913.0 Proficiency Testing 19

13.1 Enrollment 1913.2 Frequency 2013.3 Protocols for handling PT samples and results 20

14.0 Competency Assessment 2014.1 Description of method used to assess competency 2014.2 Frequency 20

15.0 Maintenance 2015.1 Pipettes 2015.2 In-house Water Purification System 2115.3 Mass Spectrometer 21

16.0 Corrective Actions 2116.1 Pre-analytic 2116.2 Analytic 2116.3 Post-analytic 21

17.0 References 21

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Quantitative Analysis of THC and Metabolites in Blood by LC-MS/MS

1.0 PrincipleThis method is used to achieve rapid and accurate low-level quantification of THC and its primary metabolites in biological specimens. This method has simple, automated sample preparation that uses isotopically-labeled internal standards. In situations of exposure to these compounds, the method would be used to identify the blood concentrations and to assist in assessing the degree of exposure.

A positive electrospray ionization method is used for the analysis. The analytes are as follows, with columns including the therapeutic ranges for the drugs in blood as given in Schulz, et. al.

Table 1. Analytes

Analyte Trade or Other Names

Blood Therapeutic Range (ng/mL)

THC Dronabinol, Marinol 5-10

THC-COOH THC metabolite n/a

THC-OH THC metabolite n/a

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 blood specimens should be collected from subjects in standard blood collection tubes.

2.3 Sample quantity

The optimal amount of specimen is at least 1.5 mL, minimum is 1.2 mL.

2.4 Unacceptable specimens

The criteria for an unacceptable specimen include low sample volumes, wrong specimen collection container, suspected contamination due to improper collection procedures or collection devices, or a leaking or damaged container. The customer is notified when a sample is rejected and further direction (i.e., submitting a new sample) is to be discussed. A description of reasons for each rejected sample should be recorded in the specimen receiving spreadsheet and/or LIMS.

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3.0 Sample handling 3.1 Specimen Identity and Integrity

All samples received will be entered into the sample receiving spreadsheet and/or LIMS. The information recorded in the sample receiving spreadsheet should at a minimum include: date sample was received in the laboratory, who received the sample, sample identification and description of sample, a description of how the sample was received (i.e. did the specimen arrive on cold packs, 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 blood samples be transported on cold packs. Special care must be taken in packing to protect the blood tubes from breakage during shipment.

3.4 Storage conditions

All blood samples should be stored at 5 ± 4 °C until analysis. Samples may reach and be maintained at ambient temperature while aliquoting and preparing for analysis; the residual specimen is again stored at 5 ± 4 °C until needed.

If the measurement is delayed until the next day, specimens should be stored at 5 ± 4 °C. If the analytical system fails, specimens and any partially-prepared / extracted samples can be stored at 5 ± 4 °C until the analytical system is again operational. Studies indicate THC is very stable when stored at varying temperatures, during freeze/thaw cycles, and in different matrices, which allows for repeat analysis.

3.5 Special equipment

Blood specimens are collected from subjects in standard blood collection tubes.

4.0 Reagents or Media4.1 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

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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 sourcesReagent Grade Source * Part

Number

Formic Acid Optima-LC/MS Fisher Scientific,Fairlawn, NJ A117-50

Type 1 Water Deionized 18 MΩ water Millipore n/a

Ammonium Formate Optima-LC/MS Fisher Scientific,Fairlawn, NJ A115-50

Methanol Optima-LC/MS Fisher Scientific,Fairlawn, NJ A456-4

Methyl tert-butyl ether (MTBE) 99.9% Acros Organics,

Fairlawn, NJ 378720025

Hexanes Optima Fisher Scientific,Fairlawn, NJ H303-4

Acetonitrile Optima-LC/MS Fisher Scientific,Fairlawn, NJ A955-4

* 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 Reagent Preparation Spreadsheet and are noted in the 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. All prepared reagents are stored at room temperature unless otherwise indicated.

4.2.1 HPLC mobile phases 100% Water with 0.1% Formic Acid (Mobile Phase A): Spike 1 mL of formic acid into 1000 mL type-1 water. This solution is good for 1 month.

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100% Acetonitrile with 0.1% Formic Acid (Mobile Phase B): Spike 1 mL of formic acid into 1000 mL acetonitrile. This solution is good for 1 month.

4.2.2 0.1% Aqueous Formic Acid Add 1000uL formic acid to 1000 mL type 1 water. This solution is good for 1 month.

4.2.3 Needle Rinse Solution – 75% MeOH Add 750 mL MeOH to 250 mL type 1 water. This solution is good for 1 month.

4.2.4 Native Standards The standards are pre-laced in wells of a 48-well plate and purchased from Cayman Chemical. The standards are stored at ≤ -20 °C. Other equivalent standards may be purchased from another vendor, if necessary, and the plates can be made by hand.

4.2.5 Isotopically labeled internal standards The labeled internal standards are pre-laced in wells of a 48-well plate and purchased from Cayman Chemical. The plates are stored at ≤ -20 °C. Other equivalent standards may be purchased from another vendor, if necessary, and the plates can be made by hand.

4.2.6 QC Material QC Material are pre-laced and dried in wells of a 48-well plate and purchased from Cayman Chemical. The plates are stored at ≤ -20 °C. Other equivalent standards may be purchased from another vendor, if necessary, and the plates can be made by hand.

4.3 Preparation of Standards

No preparation is required if using the pre-prepared standard plates from Cayman Chemical, but any time new standard materials are used, verify that the information in the instrument software reflects the actual standard concentrations and new lots of materials are verified through QC charting.

4.4 Preparation of Quality Control (QC) Materials

No preparation is required if using the pre-prepared standard plates from Cayman Chemical. Each QC should be extracted and evaluated with each run.

5.0 Test Procedure, Procedure Notes, and Safety RequirementsAn analytical batch consists of a blank, calibration standards, quality control (QC) samples, and unknown samples.

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5.1 Test Procedure

5.1.1 Sample preparation1. Print off method checklist and allow cold items to reach

ambient temperature.

2. Add 1.0mL blank blood to wells containing dried-down standards/QCs; add 1.0 mL of unknown sample to wells containing only internal standard. Mix several times via aspiration/dispensing.

3. Place plate on shaking incubator at approximately 900 rpm for approximately 15 minutes.

4. Add 0.5 mL 0.1% formic acid to all wells. Mix several times via aspiration/dispensing.

5. Place plate on shaking incubator at approximately 900 rpm for approximately 15 minutes.

6. Transfer 0.8mL of blood+acid mixture to corresponding wells of SLE+ plate.

7. Apply vacuum or positive pressure until solution penetrates well sorbent approximately 50% (or until no liquid remains on top of sorbent) – approximately 4 seconds.

8. Wait 5 minutes for sample to completely absorb.

9. Add 2.25 mL MTBE and allow to flow for 5 minutes under gravity.

10. Apply vacuum or positive pressure to complete elution – approximately 30 seconds.

11.Add 2.25 mL hexane and allow to flow for 5 minutes under gravity.

12.Apply vacuum or positive pressure to complete elution – approximately 30 seconds.

13.Evaporate to dryness in the SPE Dry at approximately 35°C under a constant flow of nitrogen. Temperatures for this step are not critical and it is appropriate to use equipment temperature readings. These temperatures do not have to be verified using NIST calibrated thermometers.

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14.Reconstitute each sample with 100 μL of methanol. Seal foil on top to prevent evaporation

15.Analyze all samples immediately or freeze at ≤ -20°C for future 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 appropriate analytical column is installed correctly (UCT Selectra DA 100 x 2.1 mm 3um)).

c. Check the needle rinse solvent level to ensure there is plenty of solvent to perform the run.

d. It is recommended to inject the solvent blank (100% MeOH) first to ensure the baseline is clean.

e. It is recommended to inject a check standard or other characterized sample that allows for chromatography and instrument performance assessment.

5.1.4 Final set-up and operation1. Check that the sample vials are in the autosampler tray. Be sure

that the autosampler chamber is set at 4°C.

2. Set up the worklist in the MassHunter software.

3. A solvent blank is generally run before the calibration curve, then the calibration curve is run, then several blanks, followed the QCs. If carryover is suspected, the Mass Hunter may choose to run a solvent blank between samples. Bracket every 20 samples with QCs and blanks.

4. Verify that the positions of samples entered on the worklist correspond to the samples in the autosampler.

5. Run worklist.

5.1.5 System Shutdown If the “standby script” is activated, the system will automatically go into Standby mode shortly after the analysis has finished.

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5.2 Equipment

5.2.1 LC-MS/MSThe LC-MS/MS analyses are performed on an Agilent 1260 quaternary liquid chromatography system (Santa Clara, CA) coupled to an Agilent 6460 tandem mass spectrometer (Santa Clara, CA).

5.2.2 Wellplate Autosampler The autosampler is configured to sample from a vial tray or two 48-well plates. A 5 second injector needle wash is performed automatically by the autosampler. The wash solvent is 75% methanol.

5.2.3 HPLC Column Oven The column oven is set at 50°C.

5.3 SuppliesSupplies should meet or exceed the listed requirements if procured from these or other sources. These are only listed as examples to aid in reordering. 5mL deepwell 48-well plates 2 mL, 12 x 32 mm, 9 mm diameter autosampler vials with the

appropriate screwcap lids Type 1 water (>18.2 Mega-ohm*cm, ultrapure) Kim-WipeTM tissues Nitrile gloves Vortexer (VWR, West Chester, PA) Thermomixer (Eppendorf, Hauppauge, NY) Pipettes, 1-mL and 100-μL (Rainin Instrument, Woburn, MA) PEEK tubing, 1/16” OD, 0.005” ID (Upchurch Scientific, Inc. Oak

Harbor, WA) PEEK Fittings, ¼-28, 1/16” (Upchurch Scientific, Inc. Oak Harbor, WA) Assorted glassware Isolute SLE+ 1mL Supported Liquid Extraction Plate (Biotage,

Charlotte, NC)

5.4 Instrument Configuration

5.4.1 Binary Pump ConfigurationThe binary pump is configured to run a gradient with premixed mobile phases with the conditions listed in Table 3.

Table 3. Binary Pump ConfigurationTime (min.) % A % B Flow Rate (μL/min.)

0 45 55 600

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3.5 45 55 600

3.6 20 80 6005.5 20 80 600

Post Run (2.5) 45 55 600

5.4.2 LC-MS/MS Configuration The LC-MS/MS configuration is described in Table 4 below. Mass spectrometer settings may vary; typical settings are shown below.

Table 4. LC-MS/MS ConfigurationParameter Setting

Column type UCT Selectra DA 100 x 2.1 mm, 3um

Mobile Phase A (0.1% formic acid in water)B (0.1% formic acid in acetonitrile)

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

Injection Volume 10 µl

Column Heater 50 ºC

Gas Temperature 320 ºC

Gas Flow 10 L/min [Nitrogen]

Nebulizer Gas 30 psi [Nitrogen]

Sheath Gas Temperature 320 ºC

Sheath Gas Flow 12 L/min [Nitrogen]

Nozzle Voltage 0 V

Capillary Voltage 3500 V

Cell Accelerator Voltage 4 V

5.4.3 QQQ configuration A LC-MS/MS (QQQ) experiment is initiated by the MassHunter software prior to the first injection. An analyte list with parent masses, fragmentation parameters, and associated product ion masses for each analyte are shown in Table 5 below.

Table 5. Analyte List with Transitions

MS Metho

dPrecurso

rProduc

t 1Produc

t 2 Analyte Fragmentor (V)

Collision Energy

(V)

Collision Energy

(V)

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(Product 1)

(Product 2)

THC 315.2 193.1 123.1 THC 136 21 23

THC 318.2 196.1 - THC-D3 132 25 -

THC 345.2 327.2 299.1 THC-COOH 128 13 17

THC 354.2 335.2 - THC-COOH-D9 136 13 -

THC 331.2 313.2 193.1 THC-OH 120 13 29

THC 334.3 316.2 - THC-OH-D3 115 9 -

5.4.4 LC-MS instrument control program An instrument control program is created using the current version of MassHunter Acquisition upon the parameters described above.

5.5 Safety

Note: All general safety precautions outlined in the Safety Manual should be followed for this method.

5.5.1 Reagent toxicity or carcinogenicity The drugs and reagents are not known to be carcinogenic. Care should be taken to avoid inhalation or dermal exposure to acetonitrile.

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 blood specimens. Follow universal precautions. Gloves, lab coats, and safety glasses must be worn while handling all human blood 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

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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 laboratory safety manual 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 laboratory safety manual for details related to specific activities, reagents, or agents.

5.5.7 Disposal of wastes Waste materials must be disposed of in compliance with laboratory, Federal, State, and Local regulations. Solvents and reagent waste should always be put in an appropriate container clearly marked for waste products. Chemical waste is disposed by an outside contractor, as needed. All disposable items that come in direct contact with the blood specimens are to be placed in a biohazard autoclave bag that should be kept in appropriate containers until sealed and autoclaved. Wipe down all surfaces with a 90%/10% methanol/water solution and allow to sit for approximately 10 min or equivalent when work is finished. All non-disposable glassware should be decontaminated with at least 90%/10% methanol/water solution for 10 minutes or equivalent before disposal, washing, or recycling.

6.0 Calibration6.1 Mass Spectrometer Calibration

A mass spectrometer checktune should be performed at least once weekly and a complete autotune should be performed at least monthly or when checktune values dictate the necessity.

6.1.1 Instrument ChecktuneThis is to be performed weekly. Place the MassHunter Data Acquisition software into the “tune” context. Allow the instrument to equilibrate and come into the ready state (denoted by a green box in the QQQ status bar). In the autotune tab, select the desired polarity for the checktune, verify the targets and tolerances are correct (see below) and click the Checktune button. The instrument will stabilize for 60 seconds before the tune begins. When

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complete, a report will appear on the screen. Save the report in the checktune folder on the desktop. If any value is unacceptable, repeat the checktune. If still unacceptable, open the appropriate tab in Manual Tune section (MS1 or MS2) and click “adjust gain & offset”. Once this is done, click on the “files” tab and save the tune. Then repeat the checktune. If still unacceptable, complete an instrument autotune.

6.1.2 Instrument AutotuneThis is to be performed monthly. Place the MassHunter Data Acquisition software into the “tune” context. Allow the instrument to equilibrate and come into the ready state (denoted by a green box in the QQQ status bar). In the autotune tab, select the desired polarity for the autotune, verify the tolerances are correct (see section 6.1.3) and click the Autotune button. The instrument will stabilize before the tune begins. When complete, a report will appear on the screen. Save the report in the autotune folder on the desktop.

6.2 Creation of standard curveStandard responses are achieved using standard referred to in Section 4.3. A linear standard curve is used and generated by taking normalizing instrument response of each analyte to the response of the internal standard.

6.2.1 Data collection A calibration curve is constructed for each run using a curve of response ratios (i.e., peak area ratio of analyte to labeled internal standard) from the calibration standards versus standard concentration divided by the internal standard concentration. All LC-MS/MS chromatograms can be smoothed. The bunching factor is adjusted as needed to achieve suitable integrations. If any standard is obviously in error, it can be removed from the calibration. No more than one standard may be discarded. If either the high or low standard is dropped, the reporting limits must be adjusted accordingly.

6.2.2 Calculation of curve statistics The slope, intercept and R-value for the calibration curve are generated using linear regression. The slope and intercept of the calibration curve are determined by linear least squares fit using the MassHunter software provided with the instrument.

6.2.3 Evaluation of curve statistics The R-squared value for the curve must be ≥ 0.99. Linearity of the standard curve should extend over the entire standard range. The intercept, calculated from the least squares fit of the data, should not

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be significantly different from 0; if it is, the source of this bias should be identified. 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, then investigations may need to be conducted to identify the source of error. If errors occur with those standards then new standards need to be prepared and evaluated along with patient specimens.

6.2.4 Calibration verification Calibration is performed as part of each analytical run and a calibration curve is constructed from the calibration standards. Additional verification is conducted by quantifying Quality Control samples of expected value against the calibration curve and statistically comparing the calculated results to expected values (i.e., %RE).

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.

7.0 Quality ControlQuality 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 calibration curve is developed for the batch using a complete set of calibration standards and evaluated as described in section 6.2. The results from the analysis of the QC material(s) obtained using this calibration curve is compared with acceptance criteria to assure the proper operation of the analysis.

7.1 Controls to be Used

Controls to be used with each sample can include a QC Low, QC Medium, and/or a QC High. These QC samples and/or a QC blank (matrix blank) are included in each analytical run and at least every 20 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 QC concentration is outside of the three-σ (standard deviation, SD) confidence interval, or if two consecutive QC concentrations are greater than 2-σ (e.g. if the first measurement is <-2-σ and the second measurement is > 2-σ).

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

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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

QC Material can be prepared using the same material that was used to build calibration standards. In this case, the QC material must be prepared on a different day. QC material can also be prepared along with standards if second source material is used. Second source material is defined as material coming from different vendors or material that has different lot numbers when coming from the same vendor.

7.3 Frequency with which Control Materials are Run

Quality controls are run after the calibration curve and before any patient samples are run. After at least every 20 patient samples and at the end of the run, the QCs are re-injected in order to ensure the instrument is functioning properly.

7.4 Establishment of Acceptance Limits for Controls

Quality Control limits will be determined during method validation studies.

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 (QualTrax).

7.5.1 Low area counts in internal referenceSignal/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.

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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 metIf 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.

7.5.3 High analyte in one standardIf an inordinately large amount of analyte is measured in one of the calibration 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 samplesIf 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. No analytical results can be reported for runs not in statistical control unless otherwise instructed by the Laboratory Director. Before beginning another analytical run, several QC materials (in the case of QC failure) or calibration verification samples (in the case of calibration failure) should be analyzed. Once calibration and/or quality control have been reestablished, analytical runs may be resumed.

7.6 Recording and Storage of QC Data

Data analysis is performed using MassHunter software as described in Section 8.0. Data are stored in both hard copy and/or electronic formats.

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8.0 Calculations8.1 Processing of data

All raw data files are quantified using the MassHunter software package. The data processing results are saved in a quantitation file that contains a numeric depiction of the retention time window, fragment ion intensities, and the area integrated for the analyte and the internal standard for each sample. The file shows the calibration curve used and its R value. The integrated peak areas for each analyte, retention times, and concentrations are saved electronically in a batch file in the quantitation software. The quantitation batch files are saved in the folder with the raw data.

9.0 Reporting Results

9.1 Reporting results to Clients

Reports are made to the client in the format requested. Results can be submitted by fax, in hard copy, or digitally. See the Quality Manual, Section 5.10, for report requirements. These reports can be automatically generated using LIMS or MassHunter. This test was developed and its performance characteristics determined by INSERT LABORATORY NAME HERE. It has not been cleared or approved by the FDA. The laboratory is regulated under CLIA as qualified to perform high-complexity testing. This test is used for clinical purposes. It should not be regarded as investigation or for research.

9.2 Range of values

The method detection limits will be determined during the validation study. The reportable range is defined as the values between the lowest quantified standard and the highest quantified standard. The initial linear ranges of the analytes are shown in table 6.

Table 6. Linear Ranges

Analyte Curve WeightingLinear Range

(ng/mL)Cut-Off (ng/mL)

THCTHC-COOH

THC-OH

9.3 Analytical Sensitivity

The LOD/LOQ will be calculated during the initial method validation to demonstrate the analytical sensitivity for reporting within our calibration

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range. Following method validation, results will be found as an appendix to this document.

9.4 Accuracy & Precision

Following method validation, accuracy and precision results will be found as an appendix to this document. The data will be continually monitored by QC charting.

9.5 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, but alteration of particular aspects of this method can result in 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.

9.6 Reference Ranges (Normal Values)

Therapeutic ranges in blood for the drugs analyzed in this method are listed (when known) in Table 1.

9.8 Critical-Call Results (“Panic” Values)

There are no critical results, but all results will be reported within acceptable turnaround time to the client.

10.0 Verification/Establishment Studies An initial method validation study for blood will be completed with at least 5 runs. Following method validation, results will be found as an appendix to this document that includes accuracy, precision, analytical sensitivity, reportable range, and interfering substances.

11.0 Package Inserts UsedNot applicable for this method.

12.0 SDS File LocationAll SDS’s are located in the laboratory and/or electronically.

13.0 Proficiency Testing13.1 Enrollment

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The laboratory will be enrolled in a PT program through an outside vendor, and enrollment is renewed annually.

13.2 Frequency

The proficiency test will be administered at least twice per year.

13.3 Protocols for handling PT samples and results

13.3.1 Sample handlingSamples are handled as patient samples. See Quality Manual, Section 5.9.3.

.13.3.2 Reporting Results

Samples are handled as patient samples. See Quality Manual, Section 5.9.3.

14.0 Competency Assessment14.1 Description of method used to assess competency

Competency is assessed by direct observation of the MassHunter performing the method, assessing problem solving skills, and reviewing data. Competency assessment form is completed during direct observation and is maintained by the QA office.

14.2 Frequency

Competency is assessed for new employees once they demonstrate 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 MaintenanceAny instrument requiring maintenance should be logged into the Instrument Maintenance Log. 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 CalibrationPipettes may be calibrated by the manufacturer and the Certificate of Analysis kept in a file for that pipette. Each pipette is verified every 6 months.

15.1.2 Corrective Action

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If delivery of pipetter does not fall within the specified tolerance limits, the pipetter must be noted by corrective action and adjusted in-house or by the manufacturer.

15.2 In-house Water Purification System

The laboratory has a Type I Water System (Millipore, or equivalent). Sanitization should be performed 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 in inoperable if the daily measured and recorded resistance is not 18.2 MΩ·cm. A corrective action must be initiated.

15.3 Mass Spectrometer

See Section 6.0

15.4 Inoperable System

In the event that a test system becomes inoperable, the vendor will be contacted.

16.0 Corrective ActionsCorrective 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 through QualTrax.

16.1 Pre-analytic

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

2. Improper storage of samples3. Failure to promptly notify submitters and laboratory section director

when a specimen is rejected4. Failure to comply with pre-analytic SOP requirements.

16.2 Analytic

1. Any QC or failure of PT due to testing errors2. Failure to comply with analytic SOP requirements

16.3 Post-analytic

1. Reporting of incorrect results2. Resampling requests caused by laboratory errors3. Failure to comply with post-analytic SOP requirements

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17.0 References Schulz M. et al. 2012. “Therapeutic and toxic blood concentrations of nearly

1,000 drugs and other xenobiotics.” Critical Care 16 (R136), pp 1-4.Quality ManualSafety Manual

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