THC-COOH CONFIRMATION



About This Document

The section supervisor and appropriate staff members review the procedures contained in this document annually. Any changes are also reviewed by the Quality Assurance Officer and the designated ASCLD-LAB Director. All Changes are signed. Old procedures are archived and retained in the laboratory for at least two years.

Table of Contents

I. Evidence Handling and Preservation

A. Specimen Handling

II. Quality Assurance

A. Equipment Maintenance and Calibration

B. Reagents, Standards and Quality Control Measures

C. Quality Control

D. Criteria for Evaluating the Internal Standards in Urine Samples

III. Reporting of Confirmatory Drug Testing

IV. GC/MS Operation and Procedures

A. Recalibration

B. Controls

C. Operation

V. Case Documentation

A. Case Notes

B. Case File

VI. Methodology

A. Initial Screening By Immunoassay

Immunochromatographic Assay Procedures

1. Principles of the Procedure

2. Quality Control

3. Operation

4. Documntation

ELISA and Procedures

1. Routine Maintenance

2. Recalibration

3. Controls

4. Operation

B. Confirmation Criteria by GC/MS

VII. Specific Analytical Procedures

A. Solvents, Solvent Mixtures, Reagents and Solutions

B. THC-COOH Confirmation

C. Basic Drug Extraction from Urine

D. Opiates in Urine

E. Cocaine and Benzoylecognine in Urine

F. Amphetamines in Urine

G. Barbiturates in Urine

H. Benzodiazepines in Urine

Appendix – A Reporting Levels

I. Evidence Handling and Preservation

SPECIMEN HANDLING

All laboratory personnel will handle submitted materials in a manner that assures the integrity of the evidence. Prior to initiating and during the processing of evidence, the analyst will employ the following practices:

The work area will be clean and free of any debris –

2. Countertops are cleaned after each case/item or when dirty

All glassware and tools to be used will be clean

Test tubes, capillary pipettes and Pasteur pipettes are used only once, then discarded

To prevent cross contamination of samples, only one case will be opened by the analyst at a time

Reagents and solvents will be kept in closed containers

During analysis the evidence will be under constant control by the analyst.

Evidence to be analyzed will be removed from evidence refrigerator and the reverse side of the pink Receipt/Request for Examination Form will be filled out.

If the subject’s name is not available at the time of log-in, the analyst will write the subjects name on the label at the time of analysis.

The analyst will ensure all identification numbers and names agree with the chain of custody receipt.

The analyst will verify and note in the case notes that the case information provided with the kit matches the HETL folder, sample information from the DRE Laboratory Drug Analysis request form submitted with the sample and all Starlims labels.

The specimen container from the collection kit will be labeled with the lab identification number, name of the subject, and the analysts initials.

A worksheet with the specimen identification number will be used and will follow the specimen throughout the analysis. Each unique case identification number will be placed onto Urine Drug Worksheet. If after initial testing the specimen is suspected to be positive for a drug, another aliquot will be used for confirmatory purposes.

After aliquoting, the samples will be assayed according to procedures noted elsewhere in this manual. Specimen jars will not be left unattended on the bench once the seals are broken. Specimens will be returned to the refrigerator.

Specimens to be confirmed by GC/MS will be retained in the refrigerator analysis is completed

Specimens, which are positive by immunoassay, will be confirmed by GC/MS procedures found elsewhere in this manual. Results of the confirmation testing will be recorded on the Urine Drug Worksheet. The worksheets, charts, chromatograms, immunoassay results, etc. will be kept in the case file.

Samples will be retained for at least six months. Chain-of-custody forms, which arrive with the specimen, will be signed by the analyst and retained with all other pertinent data in the Chemistry Office. All files will be kept in a cabinet in the Chemistry Office (Room 176) and Evidence Room (Room B-16). The file cabinet and office are locked when unoccupied.

After analysis the specimen container with the remaining urine sample will be sealed with evidence tape and the seal initialed by the analyst and placed in the appropriately labeled sample bin located in the evidence freezer located in Room 110.

The sample kit will be stored in an appropriately labeled box. This box will be retained until filled. All filled boxes will be placed in storage until returned to the submitter or destroyed.

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II. Quality Assurance

A. Equipment Maintenance and Calibration

Refer to Quality Assurance Manual

B. Reagents, Standards, and Quality Control Materials

Refer to Quality Assurance Manual

C. QUALITY CONTROL

Function checks will be performed to check the performance of equipment and agents used (either at regular intervals or while testing samples). Control checks will be performed during the analysis or testing process..

The data from the check analyses will be compared with the expected values. Any significant difference (as determined by the analyst) shall be reported to the Section Supervisor.

The following control checks will be performed during the analytical process:

Screening Testing:

• Positive controls

• Negative controls

Confirmatory Testing:

• Internal Standard

During the course of the analysis appropriate controls and standards are used to insure the validity of the analysis and that the procedure is working properly. The following measures are to be conducted within the laboratory.

Refer to the Immunochromatographic Assay, ELISA KIT and GC/MS Operation and Procedures Sections for proper quality control methods.

All results will undergo Technical and Administrative Review per the QAM

D. Criteria for Evaluating the Internal Standards in Urine Samples

Purpose of Internal Standards:

Internal standards are used to monitor extraction efficiency and instrument performance.

Evaluation of Internal Standards:

An internal standard with similar extraction, derivatization, and chromatographic properties to the analyte(s) of interest will be used.

The internal standard abundance as measured by peak area/height will be monitored for calibrators, controls and case specimens.

To maintain method reliability, the internal standard (IS) abundance in the samples should be 50% or greater than the average abundance in the most recent calibrators. Upon review of the sample, the abundance will be recorded and documented in the case file.

If it’s suspected that low abundance is due to a poor extraction, the sample will be re-extracted and re-analyzed.

If it’s suspected that low abundance is due to instrument performance, maintenance will be performed.

After re-examining the sample, if the internal standard abundance still falls below 50%:

The drug may be reported as “detected” if it has been confirmed. The reason for reporting “present” shall be documented in the case file.

This information is recorded on the Urine Quality Control Sheet (Form 119)

Reporting of Confirmatory Drug Testing

Once reviewed the result of the analysis is released for reporting. Urine Drug test results are reported in the following manner:

A. For confirmed drugs:

The following drugs were detected in this sample:

(list drugs confirmed)

or,

No drugs were detected

IV. GC/MS Operation and procedures

A. RECALIBRATION

MSD - Autotune performed daily (when in use). Relative abundance of two ions, 502amu and 219amu, must be >1% and >30% of 69amu, respectively. Failing these values, instrument is re-autotuned until values are achieved, or the instrument is put out of service. Autotune spectra are logged, dated and initialed.

B. QUALITY CONTROL

During the course of the analysis appropriate controls and standards are used to insure the validity of the analysis and that the procedure is working properly. The following measures are to be conducted within the laboratory.

i. Appropriate internal standards will be used for each extraction procedure

ii. Blanks will be run to eliminate the possibility of carryover. A blank is defined as a vial in which the previous target analyte is not detected

iii. The controls used in each analysis will be documented in the case record

C. Operation of the GC/MS

Refer to Appendix B

V. CASE DOCUMENTATION

A. CASE NOTES

The minimum information, which must be contained in the case notes are:

7. Laboratory Identification Number (Sample #)

• Analyst

Package Condition

Sample Volume

• Comments

• Suspected Drugs (if known)

• Screening Method

• Screening Results

• Extraction Procedure

• Confirmation Method

• Confirmation Result(s)

All case notes, spectra and other data generated during analysis will bear the initials of the analysts and case number.

An example of the laboratory worksheet is contained in the Quality Assurance Manual Appendix H.

B. CASE FILE

The minimal information, which must be contained in the individual case file

consists of:

8. Copy of the final report

9. Case Review Form

10. Any preliminary, supplementary or corrected reports

11. Urine Drug Worksheet

12. Evidence receipt

13. Hard copies of data that support the conclusion of the analyst.

14. Copies of reference or library spectra used for identification.

vi. Methodology

A. INITIAL SCREENING BY IMMUNOASSAY

1. RANDOX BIOCHIP ARRAY

1. PRINCIPLES OF THE PROCEDURE

The core of Biochip Array Technology is a solid state biochip onto which antibodies specific to different drug compounds are immobilized and stabilized and defined as discrete test sites. Competitive chemiluminescent immunoassays are employed for the biochip arrays. Light signal generated from each of the test regions on the biochip is simultaneously detected using digital imaging technology and compared to that from a calibration curve

2. QUALITY CONTROL

During the course of the analysis appropriate controls and standards are used to insure the validity of the analysis and that the procedure is working properly. The following measures are to be conducted within the laboratory.

i. Quality controls (both positive and negative) will be run with each batch.

ii. The controls used in each analysis will be documented in the case file

The results of all controls will be kept in the control folder

Unless approved by the vendor, expired kits will not be used for the analysis of case samples.

3. Operation

Refer to the Appendix C

INITIAL SCREENING BY IMMUNOASSAY (continued)

2. MEDTOX Immunochromatographic assay PROCEDURES

1. PRINCIPLES OF THE PROCEDURE

Assays contain a device with rapid, competitive, membrane-based immunochromatographic test strips. A single urine sample can be evaluated for the presence of specified classes of drugs in a single device. Each test strip contains antibody colloidal gold, a drug conjugate and a control line.

2. QUALITY CONTROL

An internal procedural control is included on each test strip. A line must form at the control (C) position on the test strip to indicate that adequate sample volume has been added, the reagents migrated properly, and the test strip is intact. If a control line does not form, the test is considered invalid. Formation of a visible line verifies the control line antibody antigen reaction occurred. A visible control line should always be present regardless of whether drug is absent or present.

3. Operation

1. Use test cartridge for each sample.

2. Write entire sample number on each test cartridge.

3. Follow testing protocol as stated in the manufacturer’s procedures.

4. Documentation

Take photos of each cartridge using the Use the macro setting with the flash turned-off. Turn off flash and set camera on MACRO (lighting arrow and flower)

1. Connect the camera to the computer with USB connecting cable and the USB port closest to the wall

2. Scanner and Camera Wizard window should pop up.

3. Click next

4. Choose pictures to be copied

5. Click next

6. Name should be MMDDYY for the date photos were taken

7. Pathway should be C:\OUI\MEDTOX\20—(year)

8. Check box for “delete pictures”

9. Click next

10. Photos will be downloaded and deleted

11. When prompted “what you want to do” – check “nothing”

12. Click next

13. Click “Finish”

14. When the directory window with the “just download files” highlight pops up click “Print Selected Pictures”

15. When the Photo Printing Wizard pops up click “NEXT”

16. Choose pictures to print

17. Click next

INITIAL SCREENING BY IMMUNOASSAY (continued)

18. Printer should be the HP ColorJet 2600n

19. Click next

20. Layout – Click “next”

21. After photos are sent to the printer, click “FINISH”

22. Close the pop up window

23. Place photos in corresponding case files

INITIAL SCREENING BY IMMUNOASSAY (continued)

3. ELISA OPERATION AND PROCEDURES

The ELISA Kits will be run according to the manufacturer's instructions as a minimum requirement. The routine use of the ELISA is as a Drugs of Abuse screening assay for urine.

1. ROUTINE MAINTENANCE

The Plate Reader/Washer/Shaker will be operated and maintained with quarterly system checks as a minimum requirement, as determined by the manufacturer when in use.

RECALIBRATION

All recalibration procedures will be performed as specified by the System Operation Manual. Unless earlier maintenance is warranted, quarterly maintenance will be performed as stated in the operation manual. All maintenance procedures and data will be logged in the plate reader/ washer maintenance logbook.

3. QUALITY CONTROL

During the course of the analysis appropriate controls and standards are used to insure the validity of the analysis and that the procedure is working properly. The following measures are to be conducted within the laboratory.

iii. Quality controls (both positive and negative) will be run with each batch. These will consist of controls provided with the kit.

iv. The controls used in each analysis will be documented in the case file

The results of all controls will be kept in the case file

Unless approved by the vendor, expired kits will not be used for the analysis of case samples.

4. OPERATION

Refer to the instrument’s Operators Manual

B. CONFIRMATION CRITERIA BY GC/MS

When confirming the presence of a drug in a urine/blood specimen, GC/MS will be used.

In order for a specimen to be verified as positive the retention time of the specimen must be within + 0.2 minutes of the average retention time of the controls and/or calibrators. The signal-to-noise ratio (S/N) should be at least 3:1. Quality match with library shall be 80 or greater.

VII. SPECIFIC ANALYTICAL PROCEDURES

A. Solvents, Solvent Mixtures, Reagents and Solutions

(Ref: VARIAN – Extraction of Drugs of Abuse Using Bond Elut Certify, Rev. 4.00)

1. SOLVENTS

Acetone: HPLC Grade

Acetonitrile (CH3CN): HPLC Grade

Chloroform (CHCL3): HPLC grade

Distilled Dionized Water (DIH2O): 5 ................
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