swifs toxicology laboratory procedure manual v2.2 (02.24.2009) 262 pages

8/2/2019 SWIFS Toxicology Laboratory Procedure Manual v2.2 (02.24.2009) 262 Pages

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Dallas County Institute of Forensic Sciences 8 BenzodiazepinesToxicology Laboratory Version 2.0

BENZODIAZEPINE TRAINING NOTES

1. A setting of 6 on the old evaporator gives a temperature of ~40 C.

2. Upon drying down, if a sample yields a highly oily/fatty residue DO NOT continue sampleanalysis; the sample is unsuitable for analysis. Injecting fats into the instrument will greatlydeteriorate peak shape.

3. If the instrument has not been used recently, it is recommended to ramp the GC oventemperature to 300 degrees and hold for at least ten minutes prior to the first injection to

bake out the system.

4. The SIM ions for select benzodiazepines, metabolites, and internal standards are listed below:

SIM GC/MS Methods:Mix 1 Method: BZDSIM.M Mix 2 Method: BZDSIM2.M

Oxazepam-d 5: 462, 464, 519 Oxazepam-d 5: 462, 464, 519 Oxazepam: 457, 459, 514 Flunitrazepam: 312, 286, 266Temazepam: 357, 283, 255 7-Aminoflunitrazepam: 283, 255, 254Lorazepam: 491, 493, 513 Clonazepam: 372, 374, 326Alprazolam-d 5: 284, 209, 313 7-Aminoclonazepam: 342, 344, 399Alprazolam: 279, 204, 308 Alprazolam-d 5: 284, 209, 313 -Hydroxyalprazolam: 381, 382, 383 Triazolam: 313, 238, 342

-Hydroxytriazolam: 415, 417, 380

5. Cholesterol in blood coelutes with triazolam and may preclude analysis of triazolam in blood by this method. However, urine does not typically contain cholesterol and may be a suitablespecimen for analysis in this situation.

6. Benzodiazepine metabolism varies with the subclass of drug and may occur by glucuronideconjugation, nitrogen reduction, or hydroxylation.

7. Derivatization with MTBSTFA-1%TBDMCS forms tert-butyldimethylsilyl derivatives at theactive hydrogen in hydroxyl groups, carboxylic acids, and primary and secondary amines.Compounds without active hydrogens, such as alprazolam, do not form derivatives; see

below.

This is a non-controlled copy of a controlled document


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Functional Group Derivitzation Sites:

Mix 1: Mix 2:Oxazepam amide and hydroxyl Flunitrazepam noneTemazepam hydroxyl 7-Aminoflunitrazepam none

Lorazepam amide and hydroxyl Clonazepam amideAlprazolam none 7-aminoclonazepam amide -Hydroxyalprazolam - hydroxyl Triazolam none

-Hydroxytriazolam - hydroxyl

Below is a drawing of oxazepam showing the amide and hydroxyl groups at which derivatizationoccurs. Primary amines are active sites for derivatization by silination, however, for 7-aminoflunitrazepam and 7-aminoclonazepam, derivatization does not occur at these sites. Thedecreased reactivity may be resulting from the amine being directly attached to an aromatic ring,see below.

7-AminoflunitrazepamOxazepam

Dallas County Institute of Forensic Sciences 9 BenzodiazepinesToxicology Laboratory Version 2.0



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Dallas County Institute of Forensic Sciences THC and COOH-THC by GC/MSToxicology Laboratory 1 Version 2.0

CANNABINOIDS BY GC/MS

Principle of Assay: See Cannabinoids in Urine by GC/MS for analysis of urine

Delta-9-tetrahydrocannabinol (THC) is the primary active component of marihuana. It is very lipid(fat) soluble and is rapidly distributed into body fat during the smoking of marihuana. The mostcommonly analyzed metabolite of THC is 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (COOH-THC) which is water soluble. The extraction and analysis of substances of differing solubility froman aqueous medium such as blood or urine presents an analytical challenge.

In this assay, solid phase extraction columns are used to trap THC and COOH-THC and theirdeuterated internal standards from the sample. The column is then washed with a sequence of solvents designed to sequentially remove matrix components, THC, and COOH-THC. The residuefrom the dried column eluate is derivatized with BSTFA-1%TMCS and analyzed using GC/MS withselected ion monitoring (SIM).

Equipment:

Extraction columns, such as Clean Screen by Worldwide Monitoring #CSTHC206Vacuum manifold such as Varian VacElut SPS24Hewlett-Packard model 6890 gas chromatographHewlett-Packard model 7673, 6890, or 7683 autosamplerHewlett-Packard model 5973 gas chromatograph/mass spectrometer (GC/MS)Capillary column: such as DB-1 or DB-5 (cross-linked methyl silicone gum phase) 12m to 25mConical tubes, 5 ml, screw cap with Teflon linerCulture tubes, 15 ml, 16mm x 125 mm, screw cap with Teflon linerTest tubes, 13 x 100 mmSyringes to measure standards 10 ul and 50 ul volumesCentrifugeAutosampler vials, 32 mm x 11 mm, Teflon lined seals and glass insertsVortex mixerEppendorf pipets 50 ul and 100 ul volumesRotatorHeating block for derivatizingWater bath/evaporator

Reagents:

Sodium acetateHydrochloric acid, concentratedMethanolHexaneEthyl acetate



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Acetic acid, glacialAcetonitrileBSTFA with 1%TMCS

5% Methanol in Buffer: Place 13.6g sodium acetate in a 1000 ml volumetric flask. Add water

(less than to volume), pH to 6.0 with 1N HCl, and bring to volume.Mix. Remove 50 ml of buffer from the flask and discard. Add 50 mlof methanol to the flask. Mix.

Eluants:1. THC Eluant - Hexane:ethyl acetate: Mix 95 ml hexane with 5 ml ethyl acetate.2. COOH-THC Eluant - Hexane:ethyl acetate:acetic acid: Mix 75 ml hexane, 24 ml ethyl

acetate, and 1 ml glacial acetic acid.3. Methanol:water (50:50): Mix 100 ml water and 100 ml methanol.

Safety Precautions:

The most common type of chemical or biological exposure in this type of laboratory is a splash tothe skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly usedchemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water oreye wash station. Refer to appropriate MSDSs for additional chemical information. Report theincident immediately to a supervisor. Seek medical attention as necessary.

Hydrochloric and acetic acids are corrosive. Avoid contact with skin, eyes, and mucousmembranes.

Methanol, hexane, ethyl acetate, and acetonitrile are volatile solvents. Avoid contact with skin; donot breathe vapors.

BSTFA with 1% TMCS is corrosive. Avoid contact with skin, eyes, and mucous membranes.

Stocks, Standards, Controls, Calibrators:

THC Stock Standard (1 mg/ml): Prepared by manufacturer.

COOH-THC Stock Standard (100 ug/ml): Prepared by manufacturer.

Working Mix Standard: (1000 ng/ml): Prepare two working mix standards - one for the calibratorsand one for the quality control. Transfer 10 ul THC Stock Standard and 100 ul COOH-THC Stock

Standard to a 10 ml volumetric flask and dilute to volume with methanol.

THC-D3 Stock Internal Standard (100 ug/ml): Prepared by manufacturer.

COOH-THC-D3 Stock Internal Standard (100 ug/ml): Prepared by manufacturer.



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Working Internal Standard (0.5 ug/ml):Add 50 ul of the THC-D3 Stock Internal Standard and 50 ul of the COOH-THC-D3 Stock Internal Standard to a 10 ml volumetric flask and dilute to volume with methanol.Concentration is 0.5 ug/ml for each deuterated analyte. Store in the freezer; light sensitive.

External QC Sample: Prepared by manufacturer such as Biorad.

Sample Requirements:

Acceptable specimens: blood, serum, plasma, vitreous, gastric, etc. Refer to the Cannabinoids inUrine by GC/MS procedure for analysis of urine. Note: Tissue homogenates are not suitable forthis procedure since they do not flow well through the extraction columns.

Sample Collection and Preservation:

It is recommended but not required that blood specimens are preserved with sodium fluoride (graytop) and that postmortem specimens are collected from femoral vessels. Liquids are usuallyrefrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glasscontainers often break if frozen.

Sample Preparation:

Sample dilutions must be noted in the case file.

Analytical Procedure:

Preparation of Calibrators: To 15 ml screw cap vials add the following:

2 ng/ml calibrator: Add 2 ul working mix standard to 1 ml blank blood.25 ng/ml calibrator: Add 25 ul working mix standard to 1 ml blank blood.50 ng/ml calibrator: Add 50 ul working mix standard to 1 ml blank blood.100 ng/ml calibrator: Add 100 ul working mix standard to 1 ml blank blood.

Preparation of Controls: To 15 ml screw cap vials add the following:

20 ng/ml QC control: Add 20 ul of the QC working mix standard to 1ml blank blood.Negative control: Extract 1 ml blank blood without standard added.

Preparation of Samples: To a 15 ml screw cap vial, add 1 ml of sample blood.

1. Add 50 ul working internal standard (blood) to each tube.2. Add 2 ml acetonitrile to each tube.



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3. Cap and vortex. Rotate for 10 minutes.4. Centrifuge for ~5 minutes at medium speed.5. Decant supernatant into a 15 ml culture tube and add 5 ml buffer. Vortex.6. Set up one extraction column for each specimen in the vacuum manifold. Place one 13 x 100

test tube in the collection position for each column. Secure the top on the vacuum manifold and

ensure that it is in the Waste position. Condition columns witha) 2 ml methanol (allow to flow by gravity)b) 2 ml buffer (allow to flow by gravity)

7. Add the buffer/sample mixture to the columns and allow drain by gravity.8. Rinse columns with 1 ml buffer and allow to drain by gravity.9. Dry under full vacuum for 10 minutes. Wipe out any moisture in columns with a cotton swab;

use a separate swab for each tube. Wipe bottom tips of column to remove excess water.10. Collect the THC fraction: Shift vacuum manifold lid to the Collect position. Elute each column

with 2.5 ml hexane:ethyl acetate (95:5).11. Transfer the THC fraction eluate from the collection test tube to a 5 ml conical tube and process

beginning with step 16. Return collection tube to vacuum manifold.

12. Position the manifold lid in the Waste position. Rinse columns with 5 ml methanol: water(50:50).13. Dry under full vacuum for 10 minutes. Remove excess water as in step 9.14. Position the vacuum manifold lid in the Collect position. Elute with carboxy-THC eluate with

2.5 ml hexane:ethyl acetate:acetic acid (75:24:1).15. Transfer the carboxy-THC eluate fraction to a fresh 5 ml conical tube.16. Evaporate all conical tubes in a water bath and remove immediately when dry.17. Add 50 ul BSTFA with 1% TMCS. Vortex and place in heating block at 70 oC for 30 minutes.18. Transfer to autosampler vials and analyze by GC/MS using method THC.M for THC and

COOH-THC.M for carboxy-THC.

Calculations:

The GC/MS reports the results in ug/L which must then be converted to mg/L by the chemistperforming the assay. To convert ug/L to mg/L, divide by 1000.

Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. Tocalculate the concentration of drug in the original sample, multiply by the dilution factor. Forexample if 0.5 mLs of gastric + 0.5 mL of water was analyzed instead of 1.0 mL of gastric, multiplythe calculated concentration by 1/0.5 or 2 to determine the concentration of drug in the undilutedspecimen. If the original specimen was a solid and weighed, units are mg/Kg. If the originalspecimen was a pipettable liquid, units are mg/L. Methods of dilution must be noted in the case file.

Quality Control:

A 20 ng/ml (0.02 mg/L) quality control sample and external control (Biorad) is included in each run.Results should be within range noted by manufacturer or within +/- 20% of target. QC sample results



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are maintained in the Toxicology Laboratory.

The lower limit of quantitation of the assay is 0.002 mg/L, and the assay is linear up to 0.100 mg/Lfor both analytes.

Qualifier ions should be within +/- 20% of target.

Instrument Operating Procedure:

The instrument operating procedure may be found near the instrument; refer to instrument manualand/or instrument procedure notebook.

Instrument Methods:

Refer to the Instrument Methods Notebook.

References:

Varian Applications Note: Extraction of Drugs of Abuse using Bond Elut Certify THC andCarboxy-THC in Serum, Plasma, or Whole Blood.

United Chemical Technologies Application Note: THC and Carboxy THC in Whole Blood, Serumor Plasma for GC/MS Using: 200 mg Clean Screen Extraction Column



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TETRAHYDROCANNABINOL AND METABOLITE IN BLOOD BY GC/MS

TRAINING NOTES

1. Silyl derivatives are used to improve chromatographic properties of acids, alcohols, thiols,

amines, and other functional groups containing reactive hydrogens. BSTFA forms silylderivatives of both THC and COOH-THC; see attached schematic. TMCS is present toenhance reactivity.

2. BSTFA is unstable in the presence of moisture. It is important that sources of moisture areremoved prior to derivatization examples include, drying tips of the extraction columnbefore final elutions, drying column eluates before adding derivatizing, etc. It is alsoimportant to protect the bulk derivatization reagent from moisture during storage; for thisreason, BSTFA is usually purchased and used in single use containers which are not reused.

3. It is important to notice the position of the vacuum manifold lid. The Waste position sends

column effluent into the bottom of the manifold. The Collect position sends column effluentinto the collection tube.

4. Tissue homogenates are not suitable for column extraction because they do not flow wellthrough the column.

5. Prior to this assay, specimens are usually screened by another assay such as ELISA.



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Dallas County Institute of Forensic Sciences 1 COOH-THC in Urine by GC/MSToxicology Laboratory Version 2.0

CARBOXY-TETRAHYDROCANNABINOL IN URINE BY GC/MS

Principle of Assay:

11-nor-9-carboxy-delta-9-tetrahydrocannabinol (COOH-THC or carboxy-THC) is the primary

indicator of marijuana use in the urine. COOH-THC is present in urine in the conjugated form;therefore, alkaline hydrolysis of the urine is performed first. In this assay, COOH-THC is extractedfrom urine using liquid-liquid extraction, derivatized with BSTFA-1%TMCS and analyzed usingGC/MS with selected ion monitoring (SIM).

Equipment:

Hewlett-Packard model 6890 series II gas chromatographHewlett-Packard model 5973 mass selective detectorHewlett-Packard model 7673, 6890, or 7683 autosamplerCapillary column: such as DB-1 or DB-5 (cross-linked methyl silicone gum phase) 12 m to 25 m.

Culture tubes, 13 x 15 mm, screw cap with Teflon linerConical tubes, 5 ml, screw cap with Teflon linerEppendorf pipettes - 50, 100, 200, and 400 ul volumesHamilton syringe - 50 ul volumeRotatorCentrifugeWater bath/evaporator, approximately 60 0CHeating block, 70 0CVortex mixerAutosampler vials, 32 x 11 mm, Teflon lined seals and glass insertspH paper

Wooden applicators

Reagents:

HexaneEthyl acetatePotassium hydroxide - (KOH)BSTFA with 1% TMCS (N,O-bis-(trimethylsily)trifluoroacetamide\Trimethylchlorosilane)Hydrochloric acid, concentrated10N Potassium Hydroxide: Transfer 56 grams potassium hydroxide (KOH) to a 100 ml

volumetric flask and add sufficient water to dissolve; mix. Cool and

dilute to volume with deionized water.

Hexane/Ethyl Acetate (7:1): Mix 140 ml of hexane and 20 ml ethyl acetate. Mix and store atroom temperature. Make fresh each assay.



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Safety Precautions:

The most common type of chemical or biological exposure in this type of laboratory is a splash tothe skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly usedchemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or

eye wash station. Refer to appropriate MSDSs for additional chemical information. Report theincident immediately to a supervisor. Seek medical attention as necessary.

Hydrochloric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes.

Hexane and ethyl acetate are volatile solvents. Avoid contact with skin; do not breathe vapors.

Potassium hydroxide is a corrosive base. Avoid contact with skin, eyes, and mucous membranes.

BSTFA with 1% TMCS is corrosive. Avoid contact with skin, eyes, and mucous membranes.


COOH-THC Stock Standard (100 ug/ml): Prepared by manufacturer.THC Stock Standard (100 ug/ml): Prepared by manufacturer.

Working Mix Standard: (1000 ng/ml): Prepare two working mix standards - one for the calibratorsand one for the quality control. Transfer 10 ul THC Stock Standard and 100 ul COOH-THC Stock Standard to a 10 ml volumetric flask and dilute to volume with methanol.

COOH-THC-D 3 Stock Internal Standard (100 ug/ml): Prepared by manufacturer.

THC-D 3 Stock Internal Standard (100 ug/ml): Prepared by manufacturer.

Working Internal Standard (0.5 ug/ml): Add 50 ul of the THC-D3 Stock Internal Standard and 50 ulCOOH-THC-D3 Stock Internal Standard to a 10 ml volumetric flask and dilute to volume withmethanol. Store in the freezer; light sensitive.

External QC Sample: Prepared by manufacturer such as Biorad.


Acceptable specimens: urine


Liquids are usually refrigerated during storage.



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Sample Preparation:

Sample dilutions must be noted in the case file.


Preparation of Calibrators: To a 15 ml screw cap tube add the following:

Calibrator (10 ng/ml): Add 20 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine. Vortex.Calibrator (25 ng/ml): Add 50 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine. Vortex.Calibrator (50 ng/ml): Add 100 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine.

Vortex.Calibrator (100 ng/ml): Add 200 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine.

Vortex.

Calibrator (200 ng/ml): Add 400 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine.Vortex.

Preparation of controls: To a 15 ml screw cap tube add the following:

25 ng/ml QC Control: Add 50 ul QC working mix standard (1 ug/ml) to 2.0 ml drug free urine.Vortex.

Negative Control: Extract 2.0 ml drug free urine without standard added.

Preparation of Samples: To a 15 ml screw cap tube, add 2 ml of sample urine.

1. Add 100 ul Working Internal Standard (1.0 ug/ml) to each tube with a 100 ul Eppendorf pipet. Vortex.

2. Add 200 ul of 10N KOH to each tube using an Eppendorf pipet.3. Cap tubes, vortex for a few seconds, and place capped vials in a water bath (50-60 0C) for 15

minutes. Allow to cool before proceeding.4. In the hood, add 7 drops concentrated hydrochloric acid with a disposable Pasteur pipet and

vortex to mix.5. Check the pH of each tube to ensure it is acidic. If not, add more concentrated hydrochloric

acid, vortex, and check pH again.6. Add 8 ml hexane/ethyl acetate solvent. Cap tube and rotate for about 15 minutes.

Centrifuge for about 5 minutes.

7. Transfer the organic layer (top) to a 5.0 ml screw cap conical tube and evaporate to drynessusing a water bath (50-60 0C).

8. Heat uncapped in oven at 75 deg C for about 5 minutes; allow to cool.9. Using 50 ul Eppendorf pipet, add 50 ul BSTFA-1%TMCS to each tube.10. Cap and vortex for about one minute.



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11. Derivatize in heating block at 70 0C for 30 minutes.12. Allow capped vials to cool and transfer to an autosampler vial with insert, cap and run by

GC/MS using method file THCURINE.M.

Calculations:

The GC/MS reports the results in ug/L which must then be converted to mg/L by the chemistperforming the assay. To convert ug/L to mg/L, divide by 1000.

Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. Tocalculate the concentration of drug in the original sample, multiply by the dilution factor. Forexample if 1.0 mL of urine + 1.0 mL of water was analyzed instead of 2.0 mL of urine, multiply thecalculated concentration by 2/1 or 2 to determine the concentration of drug in the undilutedspecimen. If the original specimen was a pipettable liquid, units are mg/L. Methods of dilution mustbe noted in the case file.

Quality Control:

A 25 ng/ml (0.025 mg/L) quality control sample and external control (Biorad) is included in eachrun. The results are monitored and filed for future reference. Results should be within range noted bymanufacturer or within +/- 20% of target. QC sample results are maintained in the ToxicologyLaboratory.

The lower limit of quantitation of the assay is 0.01 mg/L, and the assay is linear up to 0.200 mg/Lfor COOH-THC.

Qualifier ions should be within +/- 20% of target.

Instrument Operating Procedure:

The instrument operating procedure may be found near the instrument; refer to instrument manualand/or instrument procedure notebook.

Instrument Methods:

Refer to the Instrument Methods Notebook.



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CARBOXY-TETRAHYDROCANNABINOL IN URINE BY GC/MS

TRAINING NOTES

1. Conjugated COOH-THC is hydrolyzed by heating the urine in 10N KOH.

2. Refer to the THC blood procedure for additional training information.

3. Use reverse pipetting technique for all controls, calibrators, and standards.

4. Rinse Eppendorf pipet dispenser with deionized water after use of KOH.

5. Add HCl in hood to avoid vapors.

6. It is very important to check acidity in Step 5.

7. Ethyl acetate/hexane solvent should be made fresh each use.



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Dallas County Institute of Forensic Sciences CarboxyhemoglobinToxicology Laboratory 1 Version 2.0

CARBOXYHEMOGLOBIN

Principle of Assay:

Whole blood is treated with sodium dithionite which reduces methemoglobin to reduced

hemoglobin; methemoglobin is present in significant quantities in postmortem blood and, at highconcentration, interferes with analysis of carboxyhemoglobin (CO). This process also reducesoxyhemoglobin to reduced hemoglobin. The sample is centrifuged and aspirated into the NOVACCX. The specimen is automatically mixed with lysing agent (postmortem blood is alreadyhemolyzed), and drawn into an optical cuvette. Light from a thallium/neon hollow cathode lamppasses through the cuvette. Absorbance is measured at seven specific emission lines. Based onabsorbances at four specific wavelengths, the amount of reduced (deoxy) hemoglobin,oxyhemoglobin, carboxyhemoglobin, and methemoglobin are calculated. Turbidity andsulfhemoglobin are also detected; warnings are printed when thresholds of turbidity andsulfhemoglobin are exceeded in unsuitable samples.

Equipment :

NOVA Critical Care Xpress (NOVA CCX)CentrifugePasteur PipetsMicrocentrifuge cupsSpatulaCotton Swabs

Reagents:

Sodium dithionite (Na 2S2O4)-working bottle contents should be replaced every 6 months.Triton X-100 (p-tert-octylphenoxypolyethoxyethanol)NOVA CO-Oximeter Calibrator CartridgeNOVA CO-Oximeter Deproteinizing SolutionNOVA Controls 7, 8 & 9NOVA Total Hemoglobin (tHb) Calibrator

Preparation of Reagents:

10% Triton X-100: Measure 10 mL Triton X-100 into a 100 mL volumetric flask; Triton X-100is viscous, allow the pipette to drain. Bring to volume with deionized water. Add a stir bar and

mix for approximately 10 minutes. Allow to stand overnight before using.



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Safety Precautions:

The most common type of chemical or biological exposure in this type of laboratory is a splash tothe skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used

chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water oreye wash station. Refer to appropriate MSDSs for additional chemical information. Report theincident immediately to a supervisor. Seek medical attention as necessary.

Controls, Calibrators:

NOVA Controls 7, 8, 9


Types of samples for analysis include whole blood and blood-containing fluid from tissues such as

spleen.

Instrument Preparation:

1. Inspect sample inlet port and clean if necessary using a clean swab moistened with deionizedwater. Refer to page 3-2 in instrument manual for instructions.

2. Press SYSTEM MENU , MAINTENANCE , FLOWPATH MAINTENANCE ,DEPROTEINIZE COOX FLOWPATH buttons; follow instructions which appear on thescreen.

Calibration Procedure:

1. Verify adequate volume in the cartridges by touching the reagent graph on the top right corner of the screen. Replace any cartridges if 10% or less of the cartridge is left. To replace a cartridgepack, press SYSTEM MENU , MAINTENANCE , REPLACE/INSTALL CARTRIDGE andselect pack to be replaced. Follow the instructions on the screen.

2. To calibrate the instrument press SYSTEM MENU , CALIBRATE , INTERNAL COOXCALIBRATION .

3. External COOX Calibration (tHb Calibrator) should be performed once per month, as needed, orif the controls are repeatedly out of range using an ampoule of the Total Hemoglobin Calibrator(tHb Calibrator). To perform COOX External Calibration: press SYSTEM MENU ,CALIBRATE , EXTERNAL COOX CALIBRATION , present tHb calibrator ampoule toprobe. Perform calibration procedure 3 times. Instrument will prompt you to enter the lotnumber. Note: The complete lot number is not on the ampoule; it may be found on the tHbCalibrator box.

4. Print out the days calibration: SYSTEM MENU, CALIBRATE, DATA. Then perform one of the following:



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1) Select PRINT DIAGNOSTIC REPORT, or 2) Select COOX CALIBRATION DATA; select MENU, PRINT

Quality Control Procedures:

1. Controls are run each day the instrument is used; all three controls are analyzed. Controls arekept at room temperature.2. Press QC , Select Level: 7, 8 or 9 with proper lot number (NOT internal).3. Press ANALYZE . Present control to probe and press ANALYZE again.4. Compare results to the approved range on your print out. If a control is out of range, repeat

analysis. If a control continues to be out of range, follow troubleshooting methods in theinstrument manual.

5. If there are no problems, and the controls are within range, then the analyst may begin to runsamples.

6. Enter the QC results into the instrument maintenance log.


1. Allow specimens to come to room temperature.2. Add approximately 300 L of 10% Triton X-100 to a centrifuge cup.3. Add approximately 50 mg of sodium dithionite using a spatula.4. Add aproximately 300 L of specimen to make an approximate 1:1 dilution with the 10% Triton

X-100.5. Gently mix the zeroing solution, sodium dithionate, and specimen. Cap the centrifuge cup and

centrifuge for approximately 2 minutes.6. Remove the centrifuge cups from the centrifuge.7. Select Test Panel: COOX 8. Container should be defaulted to OTHERS .9. Enter sample information:

A. Sample Type: Postmortem Blood.B. Patient ID: Type sample name. (Ex. 05M2198 or 05H0192)

10. Press ANALYZE , present sample to probe and press ANALYZE again.11. To repeat same sample for duplicate analysis, press ANALYZE during the results screen and

press REPEAT .12. If moving on to a new sample, press NEW .13. After the last sample as been analyzed, deprotenize as described under the Instrument

Preparation Section, Step 2.

Calculations and Reporting Criteria :

Manufacturers Linear Range:Total Hb: 5-30 g/dLCarboxyhemoglobin: 0-100% saturation



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Report NOVA CCX results for % carboxyhemoglobin. Truncate to the nearest whole number.Report all concentrations below 1% as


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There are several mechanisms of death in a fire situation including exposure to elevatedconcentrations of carbon monoxide and/or cyanide and exposure to elevated temperature and fire.

Methylene chloride is metabolized, in part, to carbon monoxide. Individuals exposed to methylene

chloride may have carboxyhemoglobin concentrations in the range of 10 - 15%.

References:

Kunsman GW, Presses CL, and Rodriguez P. Carbon Monoxide Stability in Stored PostmortemBlood Samples. J Anal Tox 24:572-578, 2000.

NOVA CCX Instrument Manuals



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CARBOXYHEMOGLOBIN

TRAINING NOTES

1. Sodium dithionite may occasionally oxidize when exposed to air for a long period of time.

When this occurs, the sodium dithionite will not reduce the methemoglobin and will cause ahigh methemoglobin. Replace working sodium dithionite if this is suspected.2. The specimen for analysis must be clear of debris after centrifugation. The probe should be

placed in the center of the liquid for aspiration to prevent aspirating debris on the surface andon the bottom of the tube. A cotton swab may be used to remove fat or other debris floatingat the surface.

3. Specimen, sodium dithionite, and diluent should be mixed gently to avoid producingoxyhemoglobin.

4. Status codes and error messages should be corrected by following troubleshootingprocedures found in the instrument manual.

5. To enter a new QC lot number on the CO-Oximeter, select SYSTEM MENU, SETUP, QC.

Under the lot information tab, select ADD . Enter: lot number, level, expiration date.Select DONE . Enter the lower and upper limit for each test. Recheck entered values.Select SAVE.

6. To change the cartridge on the CO-Oximeter, select SYSTEM MENU, MAINTENANCE,REPLACE/INSTALL CARTRIDGE, CARTRIDGE #4-COOX. Follow the on-screendirections.

7. To flush the lines on the CO-Oximeter, select SYSTEM MENU, MAINTENANCE,FLOWPATH MAINTENANCE, FLUSH COOX FLOWPATH. Follow the on-screendirections.



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COCAINE AND METABOLITES by GC/MS

Principle of Assay:

Cocaine and cocaethylene are detected and may be quantitated using the Alkaline Drug Screen.

Cocaethylene is formed as a metabolite of cocaine when ethanol and cocaine are present in thebody at the same time. However, two major metabolites of cocaine ecgonine methyl ester andbenzoylecgonine are too water soluble to be extracted using the Alkaline Drug Screen. Thedetermination of these metabolites is important due to the short half life of cocaine andcocaethylene and the continued degradation of cocaine in a body after death and in vitro.

Cocaine (COC) and its metabolites cocaethylene (CE), ecgonine methyl ester (EME), andbenzoylecgonine (BE) and the corresponding deuterated compounds are extracted frombiological fluids using solid phase extraction. Sample pretreatment consists of dilution withwater and centrifugation to remove solid particulates. The supernatant is treated with phosphatebuffer and extracted using a solid phase extraction column. BE and EME present in the residue

from the dried column eluate are derivatized with pentafluoropropionic acid anhydride andpentafluoropropanol. The compounds are analyzed by gas chromatography/mass spectrometry(GC/MS) using selective ion monitoring (SIM).

Equipment:

Vacuum manifold, such as Varian VacElut SPS24Extraction columns such as Clean Screen extraction columns, Worldwide Monitoring, Cat. No.CSDAU206Hewlett-Packard model 6890 N gas chromatographHewlett-Packard model 7683 autosamplerHewlett-Packard model 5973 mass selective detectorCapillary column such as: HP-1 MS (cross-linked methyl silicone gum phase) 30m x 0.25mm x0.40um film thicknessConical tubes, 5 ml with Teflon lined screw capCulture tubes, 16mm x 125 mm with Teflon lined screw capTest tubes, 13x100 mmSyringes and pipettes to measure standardsCentrifugeAutosampler vials, 32 mm x 11 mm with Teflon lined seals and glass insertsVortex mixer1 ml serological pipettesOven or heating block for derivatizationNitrogen, compressed gas cylinderDisposable pipettesWater bath/evaporator

Reagents:

Potassium Phosphate, monobasic (KH2PO4): F.W. 136.09

Dallas County Institute of Forensic Sciences 1 Cocaine and MetabolitesToxicology Laboratory Version 2.1

This is a non-controlled copy of a controlled document.


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Sodium Hydroxide, NaOH: F.W. 40Ammonium Hydroxide (NH4OH): concentrated (14.8 M)Hydrochloric Acid (HCl): concentrated (12.1M)Pentafluoropropionic Acid Anhydride (PFAA or PFPA)Pentafluoropropanol (PFPOH)

MethanolMethylene ChlorideIsopropanolAcetonitrileEthyl Acetate

Phosphate Buffer, 0.1M, pH 6.0: Dissolve 13.6 g KH2PO4 in 800 ml deionized H20. Adjust topH 6.0 by addition of 40% NaOH. Dilute to 1000 ml using deionized H20. Mix.

Sodium Hydroxide, 40%: Dissolve 40g NaOH in deionized H20. Cool. Dilute to 100 ml withdeionized H20.

Methylene Chloride/Isopropanol/Ammonium Hydroxide (78/20/2): To prepare 200 mL, combine4 mL ammonium hydroxide, 156 mL methylene chloride, and 40 mL isopropanol. Prepare freshdaily. Mix reagents in the order listed.

Hydrochloric Acid, 0.1M: To 400 ml deionized H20 add 4.2 ml concentrated HCl. Dilute to 500ml with deionized H20. Mix.

Safety Precautions:

The most common type of chemical or biological exposure in this type of laboratory is a splashto the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonlyused chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tapwater or eye wash station. Refer to appropriate MSDSs for additional chemical information.Report the incident immediately to a supervisor. Seek medical attention as necessary.

Sodium hydroxide is a corrosive base. Avoid contact with skin, eyes, and mucous membranes.

Ammonium hydroxide is a volatile, corrosive base. Avoid contact with skin, eyes, and mucousmembranes; do not inhale vapors.

Hydrochloric and phosphoric acids are corrosive. Avoid contact with skin, eyes, and mucousmembranes.

Methanol, methylene chloride, isopropanol, acetonitrile, ethyl acetate, and pentafluoropropanolare volatile solvents. Avoid contact with skin; do not breathe vapors.

Pentafluoropropionic acid anhydride is a strong corrosive acid. Avoid contact with skin, eyes,and mucous membranes.




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Stocks, Standards, Controls, and Calibrators:

COC, CE, EME, and BE standards (1.0 mg/ml): purchase from vendor such as Cerilliant.

Corresponding deuterated standards (100 ug/ml): purchase from vendor such as Cerilliant.

Working Internal Standard Mixture (10 ug/ml): Transfer 1.00 ml of each deuterated standard to a10 ml volumetric flask. Dilute to volume with acetonitrile.

Working Standard Mixture (10 ug/ml; 50 ug/ml for BE): Transfer 100 uL of COC, CE, EME,and 500 uL of BE to a 10 ml volumetric flask. Dilute to volume with acetonitrile.

Working QC Mixture: Made the same as Working Standard Mixture but prepared independently.

External QC: purchase from vendor such as BioRad; reconstitute as noted by manufacturer;check package insert for target concentration.


Acceptable specimens: blood, serum/plasma, urine, bile, vitreous, gastric, etc.


It is recommended but not required that blood specimens are preserved with sodium fluoride(gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usuallyrefrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glasscontainers often break if frozen.

Sample Preparation:

Gastric: If gastric is a homogenous, pipettable liquid, it may be used as is in the analysis. If gastric is non-homogenous and/or thick, homogenize as tissue. For initial analysis, dilute gastric1 to 100 with water. A significant quantity of most drugs will still overload the column. If dilution is negative, an undiluted or lesser diluted sample may be run.

Other samples use 1.0 mL of sample in the analysis.

High samples: Gastric, blood, and other specimens may require dilution. In this case, extract lessthan 1 mL and make up the volume with deionized water.


1. Add 4 ml water to each 15 ml culture tube in the batch.2. Standards: Prepare 20, 100, 500, 1000, and 2000 ug/L standards of COC, CE, and EME

and 100, 500, 2500, 5000, and 10,000 ug/L standard of BE by adding 2, 10, 50, 100, and




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200 uL of Working Standard Mixture to each standard tube using a syringe or Eppendorf pipette.

3. QC: Prepare 250 ug/L COC, CE, and EME control and 1250ug/L BE control by adding25 uL of Working QC Mixture to the QC tube using a syringe.

4. Add 50 uL Internal Standard to each tube using Eppendorf pipette.

5.

Add 1 ml blank blood to the blank, each standard, and QC sample tube; add 1 ml of sample to each sample tube; add one ml of external control to tube. Vortex and let stand 5minutes.

6. Centrifuge for about 10 minutes at approximately 2000 rpm, decant supernatant into a 15mL culture tube, and add 2 mL buffer. Vortex.

7. Set up one extraction column for each specimen in the vacuum manifold. Place one13x100 mm test tube in the collection position for each column. Secure the top on thevacuum manifold and ensue that it is in the Waste position. Condition columns with:

a. 3 mL methanol (allow to flow by gravity)b. 3 mL water (allow to flow by gravity)c. 1 mL phosphate buffer (allow to flow by gravity)

8.

Add the buffer/sample mixture to the columns and allow to flow by gravity.9. Wash columns with:a. 2 mL water (allow to flow by gravity)b. 2 mL 0.1 M HCl (allow to flow by gravity)c. 2 mL methanol (allow to flow by gravity)

10. Dry under full vacuum for 10 minutes. Wipe out any moisture in the columns with aclean, cotton swab; use a separate swab for each tube. Wipe bottom tips of column toremove excess water.

11. Shift vacuum manifold lid to the Collect position. Elute each column with 3 mLmethylene chloride/isopropanol/ammonium hydroxide (78/20/2).

12. Transfer eluted samples to conical tubes and dry to residue in a water bath at about 50-55degrees C while blowing with air.

13. Heat in an oven at approximately 100 oC for about five minutes to remove any moisturefrom the tubes.

14. Let cool to room temperature then derivatize by adding 50uL pentafluoropropionic acidanhydride (PFAA or PFPA) and 50 ul pentafluoropropanol (PFPOH) to each tube; captubes; heat at approximately 70 degrees C for about 15 minutes.

15. Dry to residue in the water bath under air then reconstitute with 100 uL ethyl acetate.16. Transfer to autosampler vials and analyze by GC/MS SIM method for cocaine analysis.

Calculations:

The GC/MS calculates the results based on a 250 ug/L internal standard and reports the results inug/L which must then be converted to mg/L by the chemist performing the assay. To convertug/L to mg/L, divide by 1000.

Other Dilutions: The calculated result represents the concentration of drug in the diluted sample.To calculate the concentration of drug in the original sample, multiply by the dilution factor. Forexample if 0.5 mL of gastric + 0.5 mL of water was analyzed instead of 1.0 mL of gastric,multiply the calculated concentration by 1/0.5 or 2 to determine the concentration of drug in the




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undiluted specimen. If the original specimen was a solid and weighed, units are mg/Kg. If theoriginal specimen was a pipettable liquid, units are mg/L. Methods of dilution must be noted inthe case file.

Quality Control:

A 250 ug/L control and external control (Biorad) are included in each run; results should bewithin range noted by manufacturer or within +/- 20% of target. The sample results for cocaineand cocaethylene are also compared with the results from the alkaline screen if performed andshould be very similar.

Internal 250 ug/L QC sample should fall within +/- 20% of target.

QC sample results are maintained in the Toxicology Laboratory.

Linearity of the assay has been verified from 20 ug/L to 2000 ug/L for cocaine, ecgonine methyl

ester, and cocaethylene and from 100 ug/L to 10,000 ug/L for benzoylecgonine.Qualifier ions should be within +/- 20% of target.

Reporting Guidelines:

EME, COC, and CE: Reporting Range 0.02 - 2.00 mg/LIf result is >0.01 mg/L, but less than 0.02 mg/L and qualifiers match, report as 0.05 mg/L, but less than 0.10 mg/L and qualifiers match, report as


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References:

Varian Application Note: Extraction of Drugs of Abuse Using Bond Elut Certify

United Chemical Technologies Application Note: Cocaine and Benzoylecgonine in Serum,

Plasma, or Whole Blood for HPLC 200 mg Clean Screen Extraction Column.




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COCAINE METABOLITES by GC/MS TRAINING NOTES

1. Acetylated derivatives are produced to improve the chromatographic properties of alcohols,phenols, amines, and carboxylic acids. In this assay, benzoylecgonine and ecgonine methyl

ester are derivatized and the products of the acetylation derivatization process are describedin the Toxicology Training Manual.2. Anhydrides such as PFAA are moisture sensitive. Therefore, moisture must be dried from the

sample prior to derivatizing.3. In aqueous solution, cocaine hydrolyzes to ecgonine methyl ester. This degradation occurs in

the body after death and in vitro. Collection of specimens in gray top tubes containingsodium fluoride retards but does not eliminate the degradation process. Cocaine analysesshould be performed on blood collected in gray top tubes if available.

4. During life, cocaine is metabolized to ecgonine methyl ester primarily by action of plasmacholinesterases and benzoylecgonine primarily by non-enzymatic hydrolysis. Ecgoninemethyl ester is rapidly metabolized to ecgonine, and concentrations of ecgonine methyl ester

are usually low during life. After death, cocaine continues to break down into ecgoninemethyl ester and less to benzoylecgonine, and metabolism of ecgonine methyl ester toecgonine is slowed. Therefore ecgonine methyl ester builds up after death. A perimortemconcentration of cocaine can be estimated by converting ecgonine methyl ester present atautopsy to a molar equivalent of cocaine and adding in the cocaine concentration. Furtherinformation regarding this process is attached.

5. After the column has been cut or changed, the 500 ug/L calibration should be run as a samplein the cocscan method to determine correct windows for the EME, BE, COC, and CE.



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Dallas County Institute of Forensic Sciences CyanideToxicology Laboratory 1 Version 2.0

CYANIDE - ION SPECIFIC ELECTRODE

Principle of Assay:

The sample is mixed with acid in a Conway microdiffusion dish. Cyanide is released from thespecimen matrix, diffuses via the headspace in the Conway dish, and is trapped in a 0.1N NaOHsolution. Cyanide is quantitated in this basic solution using an ion specific electrode. Both normaland toxic concentrations can be measured by this method.

Equipment:

Conway microdiffusion dishes, with lidsVolumetric flask, 100 mLEppendorf micropipettor, 1.0 mLHamilton syringe, 50 ul

Mixer such as IKA-VIBRAX-VXR Rotating PlatformCyanide ion specific electrodeReference electrodepH/Ion specific electrode meterBeakers for electrode solutions

Reagents:

Potassium cyanideSodium hydroxideSulfuric acid, concentratedLead acetateReference electrode filling solution

0.10 N Sodium Hydroxide: Transfer 4.0 grams sodium hydroxide to a one liter volumetric flask anddilute to volume with deionized water. Store at room temperature.

6.0 N Sulfuric Acid: Add approximately 500 mL deionized water to a one liter volumetric flask andthen add 167 mL concentrated sulfuric acid. Mix and allow to cool. Dilute to volume withdeionized water. Store at room temperature.

Saturated Lead Acetate: Transfer 1.0 g lead acetate to a 10 mL volumetric flask and dilute tovolume with 1.0N sodium hydroxide. Store at room temperature.



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Safety Precautions:

Specific safety issue: Caution should be taken NOT to mix cyanide salts with an acid. Cyanide gasis given off with this reaction and the results may be fatal!

The most common type of chemical or biological exposure in this type of laboratory is a splash tothe skin or eye. Skin, mucous membranes, or eyes, which have been splashed with commonly, usedchemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water oreyewash station. Refer to appropriate MSDSs for additional chemical information. Report theincident immediately to a supervisor. Seek medical attention as necessary.

Sulfuric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes.

Sodium hydroxide is a corrosive base. Avoid contact with skin, eyes, and mucous membranes.

Biological exposure to lead may cause various health effects. Avoid accidental ingestion or

exposure; wear gloves.

Potassium cyanide can be fatal and causes multiple organ toxicity. Cyanide prevents cells fromusing oxygen. Avoid contact with eyes, skin, and mucous membranes. NOTE: Mixingpotassium cyanide and acid will release toxic cyanide gas.


Potassium Cyanide Stock Standard (1000 ug/mL): Transfer 250 mg of potassium cyanide to a 100mL volumetric flask and dilute to volume with 0.1N sodium hydroxide. Stable for 6 monthsat room temperature. Do not leave uncapped for a prolonged period of time.

Potassium Cyanide Working Standard (100 ug/mL): Prepare two working mix standards one forthe calibrators and one for the quality control. Using an Eppendorf pipet, transfer 1.0 mLcyanide stock standard (1000 ug/mL) to a 10 mL volumetric flask and dilute to volume with0.1N sodium hydroxide. Stable for one day only.


Acceptable specimens: blood, serum/plasma, urine, bile, gastric, vitreous, tissue homogenates, food,etc.


It is recommended but not required that blood specimens are preserved with sodium fluoride (graytop. For postmortem samples, femoral blood is preferable. Liquid specimens (blood, urine, etc.)



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should be refrigerated during storage; solid specimens (gastric, tissues, etc.) in plastic cups should befrozen during storage; food is usually stored refrigerated.

Cyanide concentrations are reported to vary greatly upon storage; samples should be analyzed assoon as possible.

Sample Preparation:

Tissues: Homogenize 5 grams tissue in 15.0 mL deionized water.Gastric: For initial analysis dilute 1 part gastric with 99 parts water (1 to 100 dilution). Any

significant quantity of a drug will be detected. If diluted sample is negative, anundiluted or less diluted sample may be run. Non-homogenous gastric should behomogenized prior to use.

Other samples: Dilute and homogenize as necessary. Consult a supervisor for assistance.Dilutions must be noted in the case file.

Analytical Procedure: See attached diagram of the Conway diffusion dish.

1. Prepare the 100 ug/mL potassium cyanide working standard.2. Label Conway dishes for samples,controls, and four standards (0.3, 0.5, 1.0, 2.0 mg/L).3. Fill the outside chamber of each Conway dish with approximately 1 mL of 6N sulfuric acid.4. Using a 1.0 mL Eppendorf, pipette 2.0 mL of 0.1N sodium hydroxide into the center well of

each Conway dish.5. For each standard, use a 1.0 mL Eppendorf to pipette 2.0 mL of 0.1N sodium hydroxide into

the sample chamber of the standard Conway dishes. Using a Hamilton syringe, add thefollowing amounts of potassium cyanide working standard (100 ug/ML) to the sodiumhydroxide solution in the sample chamber:

0.3 mg/L 6.0 ul0.5 mg/L 10.0 ul1.0 mg/L 20.0 ul2.0 mg/L 40.0 ul

6. Preparation of Controls: use a 1.0 mL Eppendorf to pipette 2.0 mL of 0.1N sodiumhydroxide into the sample chamber of the control Conway dishes.

1.0 mg/L QC control: Using a Hamilton syringe, add 20 uL of potassium cyanide workingstandard (100 ug/mL) to the sodium hydroxide solution in the sample chamber.Negative control: Do not add standard to the sodium hydroxide solution in the sample

chamber.

7. For each sample, pipette 2.0 mL of blood into one half of the sample chamber of the Conwaydish.



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8. Pipette 2.0 mL of 6.0N sulfuric acid into the sample chamber away from the sample orstandard. Take care not to mix the two liquids in the sample chamber.

9. Place lid on Conway dishes and rotate lid to distribute acid around the outer well to seal thedish. Carefully rotate the entire Conway dish to mix the acid with the sample or standard.

10. Place all Conway dishes on rotator platform mixer for 1 hour.

11. Approximately 1 hour before the Conway dish mixing is complete, fill the referenceelectrode with Reference Electrode Filling Solution and place the electrode in a beakercontaining 50 mL 0.10N sodium hydroxide. Add two drops of the potassium cyanideworking standard (100 ug/mL). Switch the meter to the mV setting.

12. After one hour of mixing, analyze the center well of each standard dish in sequence.Leaving the Conway dish on the mixer, remove the lid from a Conway dish and place theelectrode in the center well. (NOTE: The electrode should not touch the bottom of the dishbut should be completely immersed in the 0.1 N NaOH. Readings should be taken whilesamples are being rotated on the mixer.) After three minutes record the mV reading. Repeatthis step with each cyanide standard.

13. Place the electrode in standby, rinse it with deionized water and blot the tip with a kimwipe

before taking the next sample reading.14. Repeat Step 11 and 12 above for each sample with one additional step: after taking the first 3minute mV reading, add one drop of lead acetate solution to the center well and take asecond mV reading after 3 minutes.

15. When all analyses are complete empty reference electrode, rinse at least two times withdeionized water and blot the tip of electrode with a Kimwipe.

16. Enter the mV readings observed for the cyanide standards and the samples using the CyanideCalculation procedure found in the TOX file on the QC computer.

Calculations:

The concentration of cyanide is calculated using the Excel spreadsheet A Cyanide Calculation @ procedure found in the TOX file on the QC computer. Input the mV readings obtained for the fourstandards. Input the names and mV readings obtained for each lead acetate treated sample analyzed.The concentrations for the standards and samples will automatically be calculated and can berecorded off the screen.

To calculate the concentration of cyanide manually, use semi-log graph paper to plot mV of standards on log scale (y) versus concentration on regular scale (x). Concentration of samples isread directly from the graph using mV readings of the lead acetate treated sample.

NOTE: The results for the lead acetate treated and non-lead acetate treated sample should becompared. If results are substantially lower for the lead acetate treated sample, interference fromhydrogen sulfide should be considered. Consult a supervisor if this situation occurs.



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Dilutions: The calculated result represents the concentration of drug in the diluted sample. Tocalculate the concentration of drug in the original sample, multiply by the dilution factor.

Quality Control:

The lower limit of quantitation is 0.3 mg/L and the assay is linear up to 2 mg/L. Concentrationsoutside this range require dilution. Concentrations below 0.3 mg/L are reported as negative.

Instrument Operation:

Refer to the GHB training notes for instructions regarding operation of the pH/ion specific electrodemeter.

Interpretation:

Cyanide is found in low levels in normal, healthy individuals related to metabolism, diet, and/or

smoking. The normal value for nonsmokers is 0.01 mg/L while smokers may reach 0.05 mg/L. Thelethal concentration for cyanide is about 2.0 mg/L. A study involving 34 fatal cases showed anaverage cyanide concentration of 12.4 mg/L with the range of 1.1 - 53 mg/L.

Cyanide is generated in patients on nitroprusside therapy. Early signs of cyanide toxicity werenoticed in patients receiving nitroprusside; blood concentrations of cyanide in these individuals were0.50 - 0.65 mg/L.

Cyanide is produced during the burning of plastics and is also found in the blood of many firevictims.

Cyanide concentrations in postmortem samples have been reported to increase and decrease afterdeath due to a variety of mechanisms including bacterial action (increase and decrease) andevaporation of cyanide (decrease).

Interference:

Hydrogen sulfide, which is produced by protein decomposition, is also detected by the cyanide ionspecific electrode and may falsely elevate cyanide results in postmortem samples. Lead acetate isadded to bind the sulfide ion and reduce any sulfide interference. The difference between the samplereadings before and after addition of lead acetate provides and indication whether sulfideinterference should be considered in interpretation of results.



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References:

McAnally B, Lowry W, Oliver R, and Garriott J. Determination on Inorganic Sulfide and Cyanidein Blood Using Specific Ion Electrodes: Application to the Investigation of Hydrogen Sulfide and

Cyanide Poisoning. J. Analytical Toxicology: 3 (May/June): 111-114, 1979.

Notes:If the cyanide electrode continuously drifts and a constant mV reading cannot be obtained then theelectrode needs to be polished. The polishing procedure and materials can be found with theinstructions on how to operate the electrode.



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Conway Diffusion Dish




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CYANIDE TRAINING NOTES

Prior to analysis, rinse Conway dishes with 6 N sulfuric acid followed by deionized water.

In the event blood is not available and liver and skeletal muscle are the only tissues available,

liver should be used.



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Dallas County Institute of Forensic Sciences ElectrolytesToxicology Laboratory 1 Version 2.0

ELECTROLYTES, UREA NITROGEN, AND GLUCOSE

Principle of Assay:

Electrolytes (Na +, K+, Cl -), urea nitrogen, and glucose are determined by using the NOVA Critical

Care Express analyzer. Sodium (Na+

), potassium (K+

), and chloride (Cl-

) are measured directly byion selective electrodes. Urea nitrogen (VUN or BUN) is converted to ammonia by urease bound inthe BUN membrane. The ammonia is detected by an ammonia selective electrode; the concentrationof ammonia is proportional to the concentration of urea in the sample. Glucose is converted tohydrogen peroxide and gluconic acid by glucose oxidase bound in the glucose membrane. Hydrogenperoxide is detected by an amperometric electrode; the concentration of hydrogen peroxide isproportional to the concentration of glucose in the sample. Vitreous humor, serum, or plasma can beanalyzed for electrolytes, urea nitrogen, and/or glucose.

Equipment :

NOVA Critical Care Express (Nova CCX) analyzerSample cupsEppendorf 200 uL pipetter and disposable pipette tipsPipettor, 100 uLMicro-centrifuge and micro-centrifuge cupsCotton swabs

Reagents :

NOVA Calibrator CartridgeNOVA Chemistry Controls 4 & 5

NOVA Performance Check Solution (glucose and BUN over-range check solution)NOVA Glucose and BUN MembranesNOVA Deproteinizing Solution/ABG (Preheater Deproteinizing Solution), #12704Deionized water

Safety Precautions:

The most common type of chemical or biological exposure in this type of laboratory is a splash tothe skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly usedchemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water oreye wash station. Refer to appropriate MSDSs for additional chemical information. Report the

incident immediately to a supervisor. Seek medical attention as necessary.

Sample Requirements :

The following specimen may be analyzed by this method: vitreous humor, serum, or plasma.



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Instrument Preparation:

Inspect sample inlet port for cleanliness. If necessary, clean sample inlet port using a clean swabmoistened with deionized water. Refer to page 2-12 of the Nova CCX instrument manual for

addional information.

Calibration Procedure:

1. Verify adequate volume in the cartridges by touching the reagent graph on the top right corner of the screen. Replace any cartridges if 10% or less of the cartridge is left. To replace a cartridgepack, press SYSTEM MENU , MAINTENANCE , REPLACE/INSTALL CARTRIDGE andselect pack to be replaced. Follow the instructions on the screen.

2. To calibrate select SYSTEM MENU , CALIBRATE , ABG/CHEM CALIBRATION .Uncalibrated analytes will be crossed out at the top of the screen. If a sensor fails to calibrate, anappropriate error code is generated. If needed, refer to the troubleshooting section of the

instrument manual.3. To print the calibration report, select SYSTEM MENU , CALIBRATE, DATA , PRINT

DIAGNOSTIC REPORT . This will release the calibration report to print.

Quality Control Procedures:

1. Allow the Chemistry Perf Check Solution to thaw to room temperature (kept in freezer).2. Before opening, shake ampule for about 10 seconds.3. Select QC , Select Level: Perf Check (NOT internal). Be sure to check that lot numbers match.

Press ANALYZE . Present control to probe and press ANALYZE again to initiate aspiration.Compare results to the approved range on your print out.

4. Locate Controls 4 and 5. Thaw ampules to room temperature. Shake ampule for 10 seconds.5. Press QC , Select Level: Level 4 (NOT internal), press ANALYZE to start the analysis process.

Present Control 4 to probe and press ANALYZE again.6. Press QC , Select Level: Level 5 (NOT internal), press ANALYZE to start the analysis process.

Present Control 5 to probe and press ANALYZE again. If a control is out of range, arrows willbe next to the data indicating a high or low result. If QC is out of range, repeat analysis. If QCcontinues to be out of range, follow troubleshooting methods in the instrument manual.

7. If there are no problems, and the controls are within range, then the analyst can begin to run thesamples.

8. Enter the QC results into the instrument maintenance log.9. Check results by reading directly from the printout. If the QC samples are out of range, then the

instrument will automatically flag the result by placing a next to the test that was above the acceptable range. This may indicatea problem with the instrument; follow troubleshooting methods in the instrument manual if anerror code or status code is present.



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10. If there are no problems, and the controls are within range, then the analyst can begin to run thesamples.

11. Enter the QC results into the instrument maintenance log.12. Enter the Lot # and expiration date of all the QC samples used in the Reagent Log Book.


1. Transfer at least 500 L of the sample into a sample cup or microcentrifuge cup.2. Test panel is already defaulted to: ELECTROLYTES .3. Select container should be defaulted to OTHERS .4. Enter sample information:

A. Sample Type should be defaulted to: Vitreous.B. Type sample name. (Ex. 05M2198 or 05H0192)

5. Press ANALYZE .6. Present sample to probe and press ANALYZE again.7. To repeat same sample for duplicate analysis, press ANALYZE duing the results screen and

press REPEAT . If moving on to a new sample press NEW .8. At the conclusion of testing, pour non-diluted vitreous samples back into the appropriate morgue

tube to conserve specimen.

Calculations and Reporting Criteria :

Manufacturers Linear Range:

Analytes Serum (& Vitreous)

Sodium 80-220 mmol/L

Potassium 1.0-20.0 mmol/LChloride 50-200 mmol/L

Glucose 15-500 mg/dL

BUN 3.0-100.0 mg/dL

Results are reported as indicated by the instrument when results fall within themanufacturers linear range.

Instrument results are truncated and reported as follows:Na, Cl, glucose, and BUN truncated to a whole number (for example, 120 mmol/L)K truncated to one decimal place (for example, 3.5 mmol/L)

Results outside the manufacturers linear range are reported as greater than or less than theapplicable endpoint of the linear range.




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Results reporting should also take into consideration the following:

1. Low glucose - It is very common that postmortem vitreous humor contains less than 15mg/dL of glucose. In this situation, the NOVA will print an error message glucose low

range error. Glucose results should be reported as 100 mg/dL or a dilution with deionized water may be

performed; results must be multiplied by the dilution factor for reporting. Other analytesmust be reported from the non-diluted specimen run.

4. Low BUN If the NOVA reports BUN low range error then the sample should bereported as unsuitable for analysis.

5. Na + duplicates should vary no more than 5 mmol/L or a third sample should be analyzed.

6. Decomposed specimens may be unsuitable for analysis; if this is the case, they should bereported as such.

7. Minimum sample volume is approximately 150 ul for a single panel and 300 ul forduplicate analysis. When sufficient specimen is not available for analysis, this should benoted on the report.

8. If a sample dilution is made, multiply the instrument output by the dilution factor andnote on the instrument output.

9. Quality control samples must meet guidelines set by manufacturer. QC results aremaintained in the Toxicology Laboratory.

Instrument Operating, Maintenance, and Troubleshooting Procedure:

1. The instrument operating procedures may be found near the instrument; refer to instrumentmanual and/or instrument procedure notebook.

2. Instrument prompts to perform maintenance and warnings regarding errors in operation willappear on the Nova CRT screen. Refer to the instrument operation manual for response.



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3. The user will be prompted to change the BUN or GLU membranes approximately every 2weeks.

4. Deproteinization of the ABG (arterial blood gas) flowpath is done on an as needed basisusing the NOVA Deproteinizing Solution/ABG (also called Preheater DeproteinizingSolution). Deproteinization ruins the BUN membrane and should be performed just before

changing the BUN membrane. (Note: A more stringent cleaning of the flowpath isdescribed on page 4-17 of the NOVA instrument manual. The BUN and glucose membranesmust be replaced after this process.)

5. Instrument maintenance and repair must be documented in the NOVA InstrumentMaintenance Log.

Interpretation:

Electrolyte analysis may be used to assess hydration/dehydration status, control of diabetes,postmortem interval, etc. The following compares 2700 vitreous electrolytes performed at IFS withnormal vitreous electrolytes reported by Coe:

IFS Results of 2700 vitreous analyses performed in 1996-1997 (includes all cases, not just normals)

Sodium Potassium Chloride Glucose VUNAverage 140 9.9 123 38 21Median 142 8.6 124 0 12Std. Dev. 9.3 3.8 8.9 97.1 29.7

Coe* Normal values 10.5 29 hours postmortemSodium Potassium Chloride Glucose VUN

Average 141 8.7 118 51 18

Std. Dev. 4 1.1 7 28 8

*Reference: John I. Coe. Postmortem Chemistry, ASCP National Meeting, 1984

References:

NOVA CCX Instrument Manuals

Coe JI. Postmortem Chemistry Update. Am J Forensic Med Pathol. 14(2):91-117, 1993.



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ELECTROLYTES, UREA NITROGEN, GLUCOSE

TRAINING NOTES

1. The NOVA CCX has two flow paths: the AGB/CHEM (arterial blood gas/chemistry)

flow path and the CO-Oximeter flow path. This procedure uses the ABG/CHEM flowpath.

2. The act of removing the glucose and/or BUN sensors ruins the membranes and requiresthat the membranes be replaced.

3. If the sample contains particulate material, it should be centrifuged prior to analysis.

4. Error messages should be evaluated, and manufacturers maintenance andtroubleshooting recommendations should be followed as applicable.

5. See the instrument maintenance log for required routine maintenance.

6. Approximately three calibrators must be run before the NOVA meets calibrationspecifications.

7. Results are routinely reported as described for the following error codes:

Glucose Error 258 Glucose Low Range Error report


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UNIVERSAL ELISA METHOD Principle of Assay:

The acronym ELISA stands for Enzyme Linked Immuno-Sorbent Assay. This technique is usedas a screening procedure for opiates, carboxy-tetrahydrocannabinol, and other drugs. Positive

results from ELISA assays are confirmed using ot

swifs toxicology laboratory procedure manual v2.2 (02.24.2009) 262 pages

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