SWIFS Toxicology Resources Documents v2.0

Toxicology Resource Documents
Logbook Identification Toxicology Laboratory

Instrument Maintenance Logbook and Instrument Identification Instrument/Equipment
Agilent 6890/5973N GC/MS Agilent 6890/5973N GC/MS Agilent 6890/5973 GC/MS Agilent 6890/5973 GC/MS HP5890/5972 GC/MS Agilent 6890 GC Shimadzu GC 2010 AOC 5000 Shimadzu GC 2010 Shimadzu GC 2010 Shimadzu GCMS- QP 2010 Shimadzu, ABI LC/MS (UFLC, 3200Qtrap) Agilent, ABI LC/MS (1100 Series LC, 3200 Qtrap) Tecan Miniprep ELISA Perkin Elmer LS-5 Fluorometer Hamilton Microlab 2200 Nova CCX electrolyte analyzer Fisher AR25 pH meter Fisher 825MP pH meter Mettler Toledo AG-104 Balance Ohaus Scout II Balance Agilent 8453 UV-VIS Spec.
ID(IFS #)
0739 0161 0158 0164 0163 0160 0741/0742 0950 0951 0952 0953
Purpose
Alkaline Confirmation
Procedure included(Y/N)
Y Y Y Y Y Y Y N N N N
A/N Screen/A/N Quant/Cocaine Quant Opi, GHB, Benzo Quant THC Quant Volatiles analysis Alkaline Screening/Quant Alcohols analysis In Validation In Validation In Validation In Validation
0783
In Validation
0171 0156 0173 0740 0170 0179 0139 0140 0837
Drug Screening Salicylates SPE extractions Electrolytes and CO analysis pH buffers and solutions CN analysis Analytical Balance Top-Loader Balance In Validation
N N N Y Y Y N N N
Dallas County Institute of Forensic Sciences Toxicology Laboratory
Logbook Identification Version 2.0
Administrative Logbooks Logbook

Critical Reagent Response Factor Instrument Parameters Cleaning Security Refrigerator (X 3) DWI Return/Disposal Specimen Destruction Long Term Storage Retrieval ME Tox Cases H Cases H Cases Reciept Lab Supply/Ordering Sex Assault Kit Sex Assault Internal Chain Sex Assault Kit Returns
Purpose
Record critical reagents Record drug make-up for Response Factors Record instrument method parameters Record daily/weekly/monthly lab cleaning
Procedure included(Y/N)
Y Y N Y N N N N N N N N N N N N
Record security of laboratory and walk-in frig temp Record temperature of refrigerators Record status of completed DWI specimen Record destruction of non-DWI specimen Record retrieval from long term storage Record receipt of ME cases and requests Record receipt of H cases and requests Record information for H cases receipt Request/Order lab supplies Record received sex assault kits Hold internal chain forms Document return of kits
Logbook Identification Version 2.0
METHOD VALIDATION GUIDANCE Validation and verification of new testing methods is summarized in the Quality Manual. The Supervisor and/or Section Chief will design a specific testing protocol to validate the accuracy of a new method or to verify the accuracy of a method transferred from one instrument to another. I. Transfer of a Method to New Instrumentation First, proper calibration and operation of new instrumentation is verified and documented using the applicable instrument calibration process. Secondly, analytical results are compared between the new and old instrumentation. Where possible, side by side operation of old and new instrumentation is compared using the same samples or extracts. Standards, internal standards, and quality control samples should meet their respective criteria described in the analytical procedure. Test specimens should also be run and should meet acceptability criteria established for quality control samples or similar proficiency test results. Replicate analytical runs should be made on a single day and also compared between multiple days; criteria should meet those established for standards, internal standards, and quality controls; test specimens should meet acceptability criteria established for quality control samples or similar proficiency test results. As applicable, analysts are trained in operation of new instrumentation. II. Development of a New Analytical Method A supervisor outlines proposed steps for a new analytical method based upon literature, other established procedures, or knowledge and experience of the materials to be analyzed. The target sensitivity and linear range is established based upon expected sample conditions and instrument performance. Standard curves are run to assess the applicability of the proposed analytical technique in comparison to the targeted analytical needs. Once it is determined that sensitivity and range are appropriate, the method is evaluated for interference from expected matrices or co-occurring substances. The assay is evaluated for stability by repeating analysis of single prepared samples and duplicate preparation of samples both within day and between day. Variability of standards, internal standards, quality control samples, and specimens are evaluated with respect to literature values and/or similar established procedures. Criteria for acceptable standard curves, internal standards, and/or quality control samples are established. A formal procedure is written, applicable analysts are trained, and results are reviewed for stability within analyst. Method results may also be evaluated against external standards or specimen results from reference laboratories; criteria for acceptability will be similar to that established for quality control samples.
Method Validation Guidance Version 2.0
WORKSHEET OF POSSIBLE VALIDATION STEPS

Submitting Unit: Validation Title: Prepared by/Date: 1. Type of validation (check applicable box): Equipment Modification of an existing protocol New protocol Other: 2. Is this an original or revised validation plan? Original Revised 3. Describe the purpose or goal of the validation including the model and serial number of any new equipment: 4. Attach a copy of the written procedure: current (if applicable) and new. 5. The method is Quantitative Semi-quantitative Qualitative 6. Attach a validation plan with proposed timetable and list staff participating in the validation. The plan should lay out a course of action to assess the following, as applicable, which will be included in the final validation report: a. Method validation is the process of testing an analytical method, technique, or instrument to determine its suitability for meeting its intended purpose and to document its reliability under expected conditions of use. Generally the validation process is expected to i. Evaluate whether a new testing method meets identified analytical needs and current scientific practices; ii. Compare the new test methods performance with existing laboratory methodology; iii. Describe the conditions under which a testing method will produce valid results; iv. Predict possible sources of error; v. Determine limitations of a testing method; and vi. Establish baseline characteristics of the testing method (linearity, accuracy, etc.) which serve as benchmarks to evaluate future method
Method Validation Variables:

1. Evaluate whether a new testing method meets identified analytical needs and current scientific practices. 2. Compare the new test methods performance with existing laboratory methodology. 3. Describe the conditions under which a testing method will produce valid results. 4. Predict possible sources of error. 5. Determine limitations of a testing method. 6. Establish baseline characteristics of the testing method which will serve as benchmarks to evaluate future method performance. These characteristics may include: a. linearity/linear range: b. accuracy/precision: Accuracy is the ability to quantitate the true value of a control, standard, or sample. Precision is the closeness of agreement between many quantitations. Both accuracy and precision will be calculated using known controls and/or standards at several concentrations. The relative standard deviation and/or coefficient of variation will be used to determine precision and accuracy. c. specificity/selectivity: The selectivity of a method is the extent to which the method can determine a particular analyte of interest accurately in the presence of other components that may be expected to be present in the sample matrix. This parameter will be evaluated using actual samples and comparing the results to known methodologies. d. ruggedness/robustness: Ruggedness is the degree of reproducibility of the results obtained under a variety of conditions. These would include different analysts, reagents, extraction days, etc. These parameters are similar to precision and accuracy and will be evaluated using the results obtained from repeat analysis from several analysts. Robustness is the capacity of a method to remain unaffected by small deliberate variations in the method parameters. These changes/variations will be selected based on possible deviations from the analytical procedure such as extraction time, dry down time, solvent volume, changes in LC buffers, etc. e. repeatability/reproducibility: Repeatability is the ability of the method to accurately repeat a quantitation or identification under the same conditions (same analyst, same day, same instrument, same reagents, etc.). Reproducibility is the ability of the method to accurately repeat a quantitation or identification under different conditions (different analyst, different day, different instrument, different reagents, etc.). Each will be evaluated and reported as a relative standard deviation and/or coefficient of variation.
f. recovery efficiency: The recovery efficiency of a method is calculated by comparing the detector response to a known amount of analyte added to the sample matrix to that of a pure standard of the same quantity (concentration). This parameter will be evaluated for both compounds and expressed as a percentage recovery. g. limit of detection/quantitation: The limit of detection is the lowest concentration of an analyte in a sample that can be detected, though not necessarily quantitated. The limit of quantitation is the lowest concentration of an analyte that can be determined with acceptable precision and accuracy or is the amount equal to the lowest point on the calibration curve that can be determined with acceptable precision and accuracy. h. carryover: Carryover will be evaluated using high control samples followed by blank analysis. The high control samples will begin at the high calibration curve point and move upward through concentrations that would far exceed those expected in casework.
Example: LC/MS proposed validation study:

1. Evaluate whether a new testing method meets identified analytical needs and current scientific practices: LC/MS is in operation in many forensic toxicology laboratories and is a common technique to quantitate and identify drugs in biological specimen. The identified need in this validation is to quantitate THC and COOH-THC in blood. This will replace an existing GC/MS method in which derivatization and solid phase extraction are required. This extraction and testing method will expedite extraction and analysis time. 2. Compare the new test methods performance with existing laboratory methodology: Three analysts will compare the current GC/MS method with the LC/MS method. The same samples/site will be used where applicable. A minimum of 80 repeat samples will be run as an evaluation. LC/MS samples will be evaluated and should match closely with the results of the GC/MS assay. Any descrepancies will be reviewed. 3. Describe the conditions under which a testing method will produce valid results: A valid result will be obtained when the calibration curve is linear with a correlation coefficient (r2) of 0.995 or better, the quality control samples are acceptable ( 20% target value), the internal standard is consistent in area with the calibration sample internal standards, and the peak shapes and integrations are satisfactory upon inspection. 4. Predict possible sources of error: Some sources of error would include: matrix effects - decomposing samples, fatty samples, thin or thick blood, can cause inconsistent extraction of the drugs and unexpected ion suppression or enhancement. slight variations in solvent gradient may cause slight chromatography differences. column temperature differences may cause slight differences in elution times. synchronization of the lc injection to the mass spec data collection start may cause slight variations in elution times. a dirty pre-column filter will change peak shape. Routine maintenance will minimize this aspect. Inter-analyst variability including variance in specimen drying time. 5. Determine limitations of a testing method: This testing method is limited to testing THC and COOH-THC. This testing method is currently limited to testing blood, serum, and urine samples. 6. Establish baseline characteristics of the testing method which will serve as benchmarks to evaluate future method performance. These characteristics will consist of :
a. linearity/linear range: Calibration curves will be run using 5 calibration points. The range will be from 2 ng/ml to 100 ng/ml for both compounds. The correlation coefficient (r2) must be 0.995 (r = 0.9975) or greater to be considered satisfactory. Samples that quantitate above 100 ng/ml will be diluted appropriately and re-extracted. Samples that are detected but quantitate below 2 ng/ml will be reported as negative. b. accuracy/precision: Accuracy is the ability to quantitate the true value of a control, standard, or sample. Precision is the closeness of agreement between many quantitations. Both accuracy and precision will be calculated using known controls and/or standards at several concentrations. The relative standard deviation and/or coefficient of variation will be used to determine precision and accuracy. c. specificity/selectivity: The selectivity of a method is the extent to which the method can determine a particular analyte of interest accurately in the precence of other components that may be expected to be present in the sample matrix. This parameter will be evaluated using acutual samples and comparing the results to known methodologies. d. ruggedness/robustness: Ruggedness is the degree of reproducibility of the results obtained under a variety of conditions. These would include different analysts, reagents, extraction days, etc. These parameters are similar to precision and accuracy and will be evaluated using the results obtained from repeat analysis from several analysts. Robustness is the capacity of a method to remain unaffected by small deliberate variations in the method parameters. These changes/variations will be selected based on possible deviations from the analytical procedure such as extraction time, dry down time, solvent volume, changes in LC buffers, etc. e. repeatability/reproducibility: Repeatability is the ability of the method to accurately repeat a quantitation or identification under the same conditions (same analyst, same day, same instrument, same reagents, etc.). Reproducibility is the ability of the method to accurately repeat a quantitation or identification under different conditions (different analyst, different day, different instrument, different reagents, etc.). Each will be evaluated and reprorted as a relative standard deviation and/or coefficient of variation. f. recovery efficiency: The recovery efficiency of a method is calculated by comparing the detector response to a known amount of analyte added to the sample matrix to that of a pure standard of the same quantity (concentration). This parameter will be evaluated for both compounds and expressed as a percentage recovery. Blank blood will be used primarily, however, different types of blood such as decomp will also be evaluated. g. limit of detection/quantitation: The limit of detection is the lowest concentration of an analyte in a sample that can be detected, though not necessarily quantitated. The limit of quantitation is the lowest concentration of an analyte that can be determined with acceptable precision and accuracy or is the amount equal to the lowest point on the calibration curve that can be determined with acceptable precision and accuracy. For this
assay the limit of quantitation and the limit of detection will be defined as the lowest point on the calibration curve. h. carryover: Carryover will be evaluated using high control samples followed by blank analysis. The high control samples will begin at the high calibration curve point and move upward through concentrations that would far exceed those expected in casework. The high controls and the blanks will be carried through all parts of the extraction and instrumentation to evaluate the entire process. Individual steps may be evaluated as necessary.
Directions for Operation of the HP 6890 GC with G2070AA Chemstation

Users are referred to the appropriate instrument manuals for additional information.
There are two configurations for each instrument. One is ONLINE and one is OFFLINE. The online must be selected to run a sequence or to operate the instrument. The offline mode is to look at data or reprint reports while the instrument is in operation. The offline mode looks exactly like the online mode; however, it will not run samples or control the instrument in any way. FID Detector: FID detectors are used on the instrument. The output should be constant and stable. This may change with instrument usage or with maintenance of the detector. If the flame is out, the instrument will automatically light the flame when a method is loaded. At this time, there is no need to manually light the flame. If the flame will not light, seek assistance from a supervisor. Pausing the sequence: If the sequence must be paused during an analytical run to change an air tank or for another purpose, simply choose RUN CONTROL from the menu bar and select PAUSE SEQUENCE. When finished, resume the sequence by choosing RUN CONTROL from the menu bar and RESUME SEQUENCE. It will begin at the next line from where it stopped. The sequence does not stop immediately. It will not stop until the current sample run is complete.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Operation of the HP 6890 GC Version 2.0
Partial Sequence: Partial sequence may be used to rerun a sequence or start a sequence in the middle after stopping. This is not done after pausing it. Partial sequence may also be used in the offline mode to reprocess certain parts of a sequence in which regeneration of a report or group of reports is needed. Unless absolutely necessary, do not use the partial sequence when running a sample. Once the partial sequence has been started, samples cannot be added to the sequence; this makes the instrument unusable by other users until the sequence is complete. Starting a Run: 1. Check the gases. If low, change before starting the analytical run. If the gases run out during a run or become low during a run see the instructions for pausing a sequence. At this point a sequence must either be created or added to: A. Adding to a sequence: 1. A single click with the left mouse button on the carousel picture will bring up the sequence table. Selecting SEQUENCE from the menu bar, and then selecting SEQUENCE TABLE may also access the sequence table. 2. Using the instructions for Sequence Table, add unknown samples. B. Creating a sequence: 1. Select SEQUENCE from the menu bar and select: A. SEQUENCE PARAMETERS. Follow instructions for sequence parameters. B. SEQUENCE TABLE. Add samples using the instructions for sequence table. C. SAVE SEQUENCE AS. Save the sequence in the following format: Month (3 letters), day (2 numbers), and letter (a, b, c); for example: SEP23A 2. Go back to sequence table and select Run Sequence button to start the sequence, or select START located above carousel picture. The order of these is not important. They all must be done to properly store the data in the proper locations. The sequence will run if not saved; however, if the instrument locks up or the computer needs to be rebooted for any reason, the sequence will be lost, and sample information will need to be re-entered. Therefore, save the sequence.
2.
Operation of the HP 6890 GC Version 2.0
Sequence Parameters Screen: This screen can be accessed through the menu bar under SEQUENCE. The subdirectory is the name of the data path in which the computer will store the data accumulated from the sequence. Enter the subdirectory information in the following format: month (3 letters), day (2 numbers), and letter (a, b, c); for example, SEP23A Currently initials are not entered in the operator box. Everything else in this screen is a default value and should not be change or updated at this time. When finished simply hit the OK button to exit.
Sequence Table Screen: This screen is where the user enters the sequence to be run and sample information. This screen may be accessed by a single click of the left mouse button on the carousel picture or by selecting it under the SEQUENCE item on the menu bar. The critical information that must be present is the vial location, sample name, method name, inj/vial, and sample type. All other cells should be left blank. Any numbers in these blank cells could cause the sample to be injected improperly, calculated wrong or any number of other errors. To select a line move the cursor to the line number cell on the screen. This will produce an arrow. Click the left mouse button on this arrow and it will select the entire line (darkening the line). Drag the mouse down any number of lines to select the entire group of lines. Once a line or group of lines has been selected, the user may cut, copy, insert, insert vial range, or append line. The Insert button will insert a blank line above or in front of the line that you have selected.
The Insert Vial Range button will prompt you to enter information. Type in the appropriate information and hit the OK button. DO NOT put any number in the injection volume box. This will override the method and inject that amount, in microliters, onto the column. If left blank, it will default to the method for that information. The information that is inputted will be inserted above or in front of the line that has been selected.
The Append Line will put a blank line after the line that has been selected. The Cut button will cut the selected line. It is stored until another action takes place, i.e.: another cut or a copy. It may be pasted into another place in the sequence once it has been cut or simply discarded. The Copy button will copy the selected line but leave the line in place. It may then be pasted into another place in the sequence. The Paste button will paste the selected cut or copied line above or in front of the newly selected line. At this point, if the sequence has been saved, select the Run Sequence button to start a run. If the sequence has not been saved, select the OK button to exit this screen and save the sequence before returning. If the sequence is already running and samples are added to the sequence then simply select the OK button after the samples have been added. Then save the sequence (no need for the save as function).
Reports are printed as a sample run is completed using macros written into the method parameters. If a report must be reprinted or reprocessed, use the following instructions: Reprinting a report: If the instrument is running a sequence, use the offline mode of the instrument. From the Data Analysis screen select: Select File from the menu bar and select Load Signal 1. In the Folders: box, selected the correct data path where the signal is stored. For example, C:\hpchem\1\data\aug05c. 2. In the File name: box, selected the correct data signal to be analyzed, by clicking on the name with the left mouse button, or retype the filename of the signal desired to print. Select OK. 3. If the Integrate and print report after load button is selected, the report will be automatically generated. If it is not selected, the chromatograph can be manually integrated utilizing the Chemstation software. This will print out a formatted report. Another way to reprint a report is go into the offline mode and load the sequence for the report that you want to reprint. Select the partial sequence. At this point simply mark the line or lines that you want to regenerate and it will print the report in the proper format. This is the preferred way to reprocess a batch or reprocess more than a single sample.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Operation of the HP 6890 GC Version 2.0
Operation of the Shimadzu Headspace GC: Generating a Worklist and Runnning a Set
Double click of GCsolution desktop shortcut icon to open Operation window. Operation Manual Section 2.1.1 Single click on the 1 instrument icon. Press OK button at the Login window. When GC Real Time Analysis software is active, choose the Batch Processing icon Operation Manual Section that is in the vertical far left window. This window changes to a series of Batch icons. 3.6 3.6.3 Choose Batch Table Wizard to begin creating a worklist. Batch Table Wizard window opens select: New for Batch Table Line 1 under Batch Type C:\GCsolution\BldAlc\BldAlc.gcm is the method file then click Next Standard Locations window select: 1 for the # of sample groups Unknown Only to run QCs and cases (the most common choise) Standard Only to run a quantitative curve Standard and Unknown to run a curve followed by evaluation QCs then click Next Line1 Unknown Sample (1) window Sample Count is the total # of vials in the set Repetition is the # of injections per sample (leave set to 1) Vial # indicates the starting position of the set. The final position will automatically change depending on Sample Count and the initial vial #. Sample Name will be used to indicate which of the three autosampler trays the vial is on. Type Tray 1, Tray 2 or Tray 3. Sample ID is the ME, H-case, or DWI case number. (Leave unchanged for now.) INST Amount should be left at 1. then click Next Line1 Unknown Sample (2) window select: Create Files Automatically and Report Out Browse for the report file C:\GCsolution\BldAlc\alcreport.gcr. then click Next Summary Report window Leave Print Summary Report unselected then click Finish
A spread sheet like document will then display in the GC Real Time Analysis1 software. Change Sample IDs to appropriate case number. The Vial # column needs to be changed so that vial # 32 is not exceeded because the autosampler tray only has 32 positions. If your set has more than
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Operation Shimadzu Headspace GC Version 2.0
32 samples then change vial # 33 to 1 and re-increment (2, 3, 4, etc.) until you have the correct total number of vials. Since the second set of 1 to etc. are not on the first tray, the Tray # (Sample Name column) will need to be changed to reflect the correct tray. Do this by clicking of the spreadsheet cell and typing the appropiate Tray #. Copy and paste to make changes in other cells. Using Microsofts File Explore create a data file folder in C:\GCsolution\InstA or Bdata\currentyeardata. Return to the GCsolution software and right click on the Data File column and choose settings from the drop-down menu that appears. Select: Folder tab. Use Specified Folder De-select: Use the same folder Select the folder just made for Data File. Select the Data Filename tab. Create Files Automatically should already be selected. If not, do so at this time. In the Selected Items: window make sure that only sample ID is present. De-select all other options. then click OK. Save the batch file. Under the file drop-down menu select Save Batch File as. Save the batch file in C:\GCsolution\Sequence\currentyearSequence.
Create a job(s) on the autosampler to run your set. This is done using the Control Terminal for the autosampler, not the key pad for the GC. Select: Add Job (F2) Append appropriate Tray, Vial info., and method (Bldalc) Home (F4) If the set contains more than 32 vials, a second job on another tray will have to be generated to accommidate the additional vials. To start the run select the Start Button icon in the GC Real Time Analysis software. When the software displays Ready(Standby), select the correct Job on the autosampler and press Start (F4).
Adding a batch to the Job Queue

Open the offline editor for instrument 1. It is the document looking icon positioned Operation Manual Section just below the Instrument 1 icon on the Operation window that appeared at the 2.1.1 & 7.3.2 start of operation. Proceed with making a Batch File as is normally done. After saving the batch file, switch to the online software. Under the Batch drop-down menu, select Batch Queue. Select the Add button, find the desired batch file, select open, then OK button. An autosampler Job also needs to be appended to the autosampler. Create this job as is normally done.
Operation Shimadzu Headspace GC Version 2.0
Calibrating Via a Multi-point Curve Operation Manual Section 2.1.1 & 7.3.2 Create standardization curve points following the Alcohol procedure. Also prepare QCs
to evaluate the new calibration. Enter the Batch Table Wizard Batch Table Wizard window opens select: New for Batch Table Line 1 under Batch Type C:\GCsolution\BldAlc\BldAlc.gcm is the method file then click Next Standard Locations window select: 1 for the # of sample groups Standard and Unknown to run a curve followed by evaluation QCs then click Next Line1 Standard Sample (1) window select: 5 for Calibration Levels 1 for repetition enter the appropiate vial starting #. Enter the appropiate tray in the Sample Name column Sample ID will be the curve point identifier Then click Next Line1 Standard Sample (2) window select: Create Files Automatically Report Out C:\GCsolution\BldAlc\alcreport.gcr Then click Next The following windows and actions will be identical to setting up a run of cases. In this instance the setup will be for the QCs that evaluate the new calibration curve. Following the completion of the calibration run, a QC Report for one of the evaluation QCs should be printed and stored. The r2 value must meet current lab requirements.
Operation Reprinting Alcohol Reports and QC Reports Manual Section 2.1.1 & 7.3.2 Click on the Post Run icon on the Operation window. Select the Report Generator
icon from the far left window. If this icon is not present click the Top arrow icon. Under the drop-down file menu choose: open format file alcreport.gcr is for regular alcohol data - qc report.gcr is for printing calibration curves and r2 values load data file choose the file to be printed print
Dallas County Institute of Forensic Sciences Toxicology Laboratory Operation Shimadzu Headspace GC Version 2.0
This is a non-controlled copy of a controlled document
THERMOMETER CALIBRATION CHECK 1. Annual Calibration Check 1.1. Reference Thermometers 1.1.1. NIST traceable reference thermometers are maintained under the direction of the Quality Manager. 1.1.2. Calibration of appropriate reference thermometers is verified annually by an outside vendor. 1.1.3. Records are maintained by the Quality Manager. 1.2. General Use Thermometers 1.2.1. Calibration of laboratory mercury and alcohol thermometers will be verified annually against a NIST traceable thermometer. 1.2.2. General use thermometers should read within +/- 2 divisions of the NIST thermometer. 1.2.2.1. If the general use thermometer does not meet the designated range, it will be replaced or repaired. 1.2.2.2. In the event that replacement or repair is not an option, the amount of deviation from the NIST thermometer is recorded on applicable log sheets and laboratory thermometer readings are adjusted accordingly. 1.2.3. Records of annual thermometer calibration checks will be maintained in the laboratory.
Thermometer Calibration Check Version 2.0
Operation of the 5973 Series Mass Spectrometer

Utilizing G1701BA and G1701DA, Versions B.01.00 and D00.00 Mass Spectrometer Chemstation Software Overview The Chemstation software is a windows based application and usable on Windows 95, 98, NT and 2000. The following instructions are intended as a general guide and do not represent all of the ways that the software may be used. In depth literature is published by the manufacturer and should be used as the key reference and troubleshooting guide. Online help is also available at www.agilent.com. Users may begin using the Chemstation software by clicking on the Top icon located on the desktop. There are several ways to move around in the Chemstation software. The primary method of movement from one screen to another in the software is through the View menu item. If in the Top Level, Instrument Control or Data Analysis, the View menu item will allow the user to move to any of the other screens. Users are referred to the appropriate instrument manuals for additional information. Top Level Screen From the Top Level screen the user has the ability to edit, print, load, and save methods and sequences. This screen enables the user to begin instrument operation. Instrument Control Screen From the instrument control screen the user has the ability to modify instrument parameters such as injector, inlet, column and oven parameters, mass spec temperatures, and electron multiplier voltages. The user can also load, edit, save and print methods from this screen. This screen enables the user to monitor instrument parameters during a run. Instrument tuning is also done from this screen. Data Analysis Screen From the data analysis screen the user can edit the data analysis portion of a method, edit and select libraries, review total ion chromatograms, review individual spectra, compare unknown spectra to known libraries, and print reports. Data analysis is also the location of the quantitation databases used in quantitative methods. Storage of Data files, Methods and Sequences The hard drive is partitioned into two drives, drive C:\ and drive D:\. The C:\ drive is the location of the Chemstation and the operating system (Windows NT 4.0 or Windows 2000). The D:\ drive is designated for storage of other installed software. For the operation of the Mass Spectrometer Chemstation there are three key areas of storage.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Operation 5973 GC/MS Version 2.0
Data is stored in C:\hpchem\1\data\ or C:\msdchem\1\data\. Data consists of data.ms files containing total ion chromatograms and their spectra, pre_post.ini files containing information on the status of the instrument during the run of the indicated file, and log files containing information regarding the sequence that was run. Methods are stored in C:\hpchem\1\methods\ or C:\msdchem\1\methods\. Methods contain all of the information required to execute a run. Methods contain the required macros and instrument parameters such as oven ramping, pressure and flow parameters, injection temperatures and specifications, and instrument temperatures, as well as other information. Sequences are stored in C:\hpchem\1\sequence\ or C:\msdchem\1\sequence\. Sequences contain the information required to complete a series of injections utilizing the autosampler tower, tray and barcode reader. The information consists of vial number, sequence line number, sample name, method name, and operator information.
I. Sequences Loading a sequence or creating a new sequence From the top level: 1. Select Sequence menu item. 2. Select Load menu item. 3. Select the default.s or a previous sequence and OK. This sequence of events will load the selected sequence in the instruments memory. Proceed to Edit sequence section to edit the sequence or Running a sequence section to run the loaded sequence. Edit sequence Once the desired sequence has been loaded you may want to edit the sequence. From the top level: 1. Select Sequence menu item. 2. Select Edit Sample Log Table menu item. The edit screen will appear at this time with the sequence that the user had previously loaded. The user may edit, cut, copy, paste, or repeat lines in the sequence. Information that is required is Type, Vial, Method, and Expected Barcode. Line and Data File will be completed automatically by the Chemstation software. The user may wish to include a Sample Name and/or Miscellaneous Information.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 2 Operation 5973 GC/MS Version 2.0
Line the line number in the sequence completed by the Chemstation Type the type of sample: sample, blank, calibrator, etc. Data File unique number for a particular sequence generated by the Chemstation. This number is only unique for the specific sequence and may be repeated in other sequences. Method the method that the user wishes to employ. Sample Name the description of the sample. Repeat will increment the highlighted line by one number and will copy the highlighted line for all other information. Cut will delete the highlighted line (will hold it in memory for paste function until another cut or a copy is executed). Copy will copy the highlighted line to be used with the paste function. Paste will paste the highlighted line that has been cut or copied and insert it above the current highlighted line. Once the editing is complete select OK to exit. If you do not exit out of the edit screen the sequence will not continue.
Edit a sequence while it is running The user may also edit the sequence while a sequence is running to add specimen to the running sequence. If the sequence is running the menu items will not be available and the user must select the Edit Samp Log Tbl button from the center screen. At this time the edit sequence screen will appear.
Saving a sequence After the user has loaded and edited a sequence, the sequence must be saved. From the top level: 1. Select the Sequence menu item. 2. Select the Save menu item. The default location to save sequences is C:\hpchem\1\sequence\ or C:\msdchem\1\sequence\. If the location varies from the default location, locate the default and then save. The format in which to save the sequence is: month, day, and letter designation (ie: Jul29A).
Running a sequence There are several ways to begin a sequence. The user may select Run, Load and Run, or Position and Run. The Run menu item will begin the current sequence. The Load and Run sequence menu item will allow the user to load a new sequence
and then run it. The Position and Runmenu item will allow the user to begin the current sequence at a user specified location within the current sequence. To begin a sequence from the top level: 1. Select the Sequence menu item. 2. Select the Run, Load and Run, or Position and Run menu item. 3. The user will be prompted to enter the data path for the storage of the data collected during the sequence run. The following screen will appear:
4. Make sure that Full Method and Inject Anyway are selected. The user may or may not want to select the Overwrite Existing Data Files option. 5. Type the correct path for the storage of data. The default path is C:\hpchem\1\data\ or C:\msdchem\1\data\. The default location is where the Chemstation looks for data from the data analysis screen. If the user does not store the data in the default location be aware of the location that was selected. 6. Select Run Sequence button. If the sequence does not begin at this time, check the above screen and the selections to make sure that the proper selections have been made. If Load and Run or Position and Run are selected the user will be prompted to load a sequence or select a position within a sequence before the above screen appears. Using Keywords The Chemstation allows the use of keywords within a sequence. These keywords allow the user to perform certain functions within the sequence such as running a command or macro, tuning the instrument, or pausing the sequence. The most common keyword used is Pause. The Pause command will allow the user to pause the sequence after an injection or series of injections are made, or a method is complete. To use a keyword:
1. From the Sample Log Table: 2. Select the next line to be run, copy and paste so there are two identical lines. 3. Select the Keyword command in the Type box. 4. This will prompt the user to type the Keyword that is to be used. Select the keyword desired in the keyword box. 5. Select OK. When the current method is complete the keyword command will be executed.
II.
Methods
The method in the Chemstation software is the location of all of the instrument parameters and data analysis parameters that will take place during the execution of a method in a sequence. Methods should only be created or modified by those with knowledge of the required parameters or experience with instrument parameters. Creating a new method To create a new method the user may select menu items from either the Top Level screen or the instrument control screen. From either screen: 1. Select the Method menu item. 2. Select the Load menu item. 3. Load the default method or load an existing method and modify to create your new method. 4. Select the Save menu item to save the method with an appropriate name.
Operation 5973 GC/MS Version 2.0
Editing an existing method If the user needs to edit a method or check the contents of a method the method may be accessed from the Top Level screen or the instrument control screen. From either screen: 1. Select the Method menu item. 2. Select the Edit Entire Method menu item. 3. Edit or review the method. Save the method ONLY if aware of the changes that have been made. DO NOT save the method if unaware of the changes made.
III. Data Analysis The user may enter data analysis in several different ways. The user may open it from the icon on the desktop, from the Top Level screen during the execution of a sequence, or from the View menu item from any screen.
Loading a chromatogram (data file) The data file contains the total ion chromatogram and the spectra for the identified compounds in the chromatogram. To load a chromatogram: 1. Select the File menu item. 2. Select the Load Date File menu item. 3. Select the appropriate data file from the correct data path and select OK. (The data file will be in the location that was specified when the sequence was started. The default location is C:\hpchem\1\data\ or C:\msdchem\1\data\.)
Reviewing Data Once the chromatogram has been loaded it will appear in window [2]. It will appear as the total ion chromatogram (TIC) with peaks representing detector response to compounds and their retention times. There are many items that the user may want to use to review data. These include, but are not limited to, integrating, searching for specific ions, comparing unknown spectra to known libraries, generating reports, and printing spectra. The TIC will be normalized on the largest peak. This means that the largest peak will be completely visible from the top of the peak to the baseline. Large peaks in a TIC may make smaller peaks not visible when looking at the normalized chromatogram. If the user wishes to look at the smaller peaks the zoom function may be used. To do this left click and hold the mouse, drag a box around the area to be analyzed and release. This will zoom in on the area that the box was drawn around. Generating, comparing and printing spectra To generate a spectra at a specific TIC retention time double click the right mouse button at the point of interest. This will generate a mass spectrum of the area that was clicked on and it will appear below the TIC in window [1].
The method that is loaded will determine what library or libraries have been selected for use (see searching libraries for instructions on changing). To compare the generated spectrum with the library that is specified in the method: 1. Double click the right mouse button while the cursor is in window [1].
2. This will generate a comparative spectrum with the best match being shown first. This comparative spectrum is generated in window [24].
The spectrum for the unknown compound is located on top and the spectrum for the best match is located on the bottom. The library that the best match spectrum came from is listed in the PBM Search Results box. 3. To print the comparison, select the Print button located in the PBM Search Results box, or select the File menu item, Print and Selected Window. Type in the number 24 when prompted. The user may also want to print the TIC or the unknown spectrum without using the comparison method. To do this: 1. 2. 3. 4. Load chromatogram and generate spectrum. Select the File menu item. Select the Print menu item. The user will be prompted to enter the window to print. Enter the number of the window desired to print: 1 = spectrum, 2 = TIC.
Searching for specific ions When a specific ion needs to be isolated so the user may separate coeluting compounds or find a compound with a weak response, the Chemstation can search for specific ions within a TIC. To do this: 1. Select the Chromatogram menu item. 2. Select the Extract Ion Chromatograms menu item 3. This will prompt the user to enter the ion or ions that the Chemstation will look for and the retention time windows in which to look. 4. Enter the ions of interest. 5. Enter the retention time window in which to look. 6. Select the OK button. This will prompt a window displaying the extracted TIC and each ion will be displayed in a different color. Full spectrum may be generated by double clicking the right mouse button on the area of interest. It may be of value to the user to overlay these extracted ion chromatographs. To do this, select the Chromatogram menu item and Display Ion Chromatogram in Merged Format. Subtracting Spectra In the event that two compounds coelute or a compound with a weak response is hidden in the baseline it may be desirable to subtract interfering spectra. An example would be a drug that gives a weak response and is hidden in the baseline. In this instance the user would look for the ions in the compound of interest by a manual search or by searching for extracted ions. Once the compound is found the full spectrum may have ions from the baseline included in the ions for the compound. The Chemstation allows the user to subtract one spectrum from another to clean up or clarify a spectrum. To subtract a spectrum: 1. Obtain a mass spectrum of the desired compound (with the interfering ions). 2. Move the cursor to a location, usually just before or just after the compound of interest, and obtain a spectrum of that area. 3. Select Spectrum menu item. 4. Select Subtract menu item.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Operation 5973 GC/MS Version 2.0
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The resulting spectrum will be the difference of the second obtained spectrum removed from the first obtained spectrum. Printing reports There are many ways to print reports and a number of reports are available in the Chemstation software. The most common way to print a report for a designated method is to: 1. Load the appropriate TIC. 2. Select the Method menu item. 3. Select the Run Method menu item. 4. This will run the data analysis portion of the method that has been selected in data analysis. It will print the report as if the sample had been run in a sequence. The user may also want to use the options available under the quantitate and tools menu items.
Using the Quantitate and Tools menu items the user will be able to use the Chemstation report format and to reprocess quantitative and qualitative reports linked to custom reports. Searching Libraries There are many different libraries that are available in the Chemstation ranging from inhouse libraries to commercially purchased libraries. The libraries are located on the C:\ drive of the computer in C:\database. To change the libraries that are automatically searched in a method:
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1. Select the Spectrum menu item. 2. Select the Select Library menu item. 3. This will prompt the user to type in the name of the library to be used. This will affect the library that is used when the user is comparing an unknown spectrum to a library spectrum. Note: Mass Spectral libraries are used only as a tool in identifying an unknown. Chemists must consider a variety of factors before reaching a conclusion.
Addition of New Mass Spectra to Libraries If a new compound or standard is obtained, it is important to have this compounds mass spectra in a spectral library. The spectral library to place the new compound must be selected, usually it will be dcflab.l. (See instructions above to select the appropriate library.) To add a mass spectrum to a library: 1. Select the Spectrum menu item. 2. Select the Edit Library menu item. 3. Select the Add New Entry menu item.
4. Enter information into appropriate cells: Name, Mol. Formula, Mol. Weight, etc. 5. Select the Include in search box.
6. After entering the information, select OK, and the Chemstation software will add the spectra into the library in the C:\database\ file path. 7. The Chemstation software will also assign the new spectra a number.
Parametric Retrieval is located under the View menu item in Data Analysis screen only. Parametric retrieval can be used to search a user designated library for compounds based upon name, molecular weight, CAS number, library entry number, etc. However, the information must be in the library before the search parameters will work. Occasionally all of the information may not be available with in-house libraries.
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Tuning the 5973 The tuning instructions and parameters are located in the maintenance log and the reference collection found near each instrument.
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Gas Chromatography CALIBRATION AND MAINTENANCE Agilent/HP 6890 Gas Chromatograph Coupled with Flame Ionization Detectors All chemists are responsible for ensuring the proper operation of gas chromatographs (GC). A chemist is assigned primary responsibility for overseeing maintenance and troubleshooting of the GCs. All chemists are responsible for running QC samples, evaluating instrument calibration, and providing general housekeeping of instrumentation as needed. I. Instrument Calibration:
Calibration of the GC will be evaluated each day that the GC is used. This will be accomplished by running quality control samples (QC). If the QC sample does not meet set criteria, the following steps will be performed until acceptable results are obtained: rerun QC, re-extract and rerun QC, make new stock with re-extraction and reanalysis, recalibrate with successful analysis of a new QC sample, and/or seek supervisory assistance. Additional information may be found in the appropriate analytical procedure. Calibration of retention index (RI) will also be evaluated for these drugs and internal standard each day the GC is used. Retention index is properly calibrated when these drugs fall into the expected retention index window established in the GC method. Based upon the retention time of known standards, the Calibration Table for the drug and/or internal standard will be updated, the method saved, and the chromatogram reprocessed as necessary. A supervisor will be notified if appropriate or if problems persist. II. Instrument Maintenance:
Routine preventive maintenance such as changing a gas cylinder, septum, and/or inlet liner is noted GC Maintenance Log located near each GC. Scheduled preventive maintenance is noted below. Activities other than scheduled preventive maintenance should be authorized in advance.
GC Maintenance 6890 Version 2.0
GC Maintenance Schedule Daily/with use Bi-Weekly As needed Change wash vial solvents Check gases Change septum Change liner and o-ring Trim column Change gold seal Change gas tanks Replace column Clean split side arm Clean FID and jet
Maintenance activities should be consolidated to minimize instrument down time. For example, if it is time to change the gold seal and cut the column, it may be more prudent to change the liner and septum at that time or hold off changing the gold seal until the liner and septum are changed. The maintenance interval is not a rigid schedule and should be based upon workflow. Unless there is an immediate problem, a chromatographic run should continue to completion, and the maintenance performed at the end of a run. Where duplicate instrumentation exists, only one instrument at a time should be scheduled for preventive maintenance to avoid multiple instruments being out of service at the same time. Ultraclean Technique: Liners, gold seals, and certain other parts must be handled using ultraclean techniques to avoid contamination of the part with oils from the skin, plasticizers from plastic laboratory benchcoats, etc. Do not handle an ultraclean part with your hands; wear cotton gloves or use a fresh Kimwipe. Lay the ultraclean part on its cloth wrapping or on a clean Kimwipe. Changing the Wash Vial Solvent: Discard waste solvent into the hazardous waste. Replace spent solvent in wash vials with fresh methanol, and replace the vials in the appropriate location on the instrument autosampler. Changing the Liner and Septum: Tools needed: inlet wrench and tweezers. NOTE: Turn off carrier gas; if the septum nut is removed without turning off the carrier gas, the glass wool plug may be blown out.
Use ultraclean technique at all times when handling the new liner. 1. Cool the inlet to 80oC to minimize oxidation and prevent burns. 2. Turn off the carrier gas on the GC by using the software located on the front panel on the GC: From the front panel on the GC, push the FRONT INLET button. Scroll down using the button to the Total Flow line. Turn off the flow by pressing the OFF button. Once maintenance is complete, turn the flow back on by pressing the ON button. 3. Once the inlet is cool and the gas is off, remove the top septum nut. Remove and replace the septum. Reinstall and tighten the top septum nut. These are not ultraclean parts but care should be taken to avoid any unnecessary contamination. 4. Remove the nut covering the liner. Remove the old liner and discard. 5. Using ultraclean technique, carefully slide an o-ring over the end of the new liner, approximately from the top of the liner, and slide into the inlet. The glasswool plug, if present, should be nearest the bottom of the liner. 6. Replace and tighten the nut and turn on the inlet temperature and carrier gas flow. 7. A blank should be run to bake out the new liner before analyzing samples. Changing the Gold Seal: Tools needed: appropriate screw driver (Phillips/Torx/etc.), 9/16 wrench, wrench Note: The gold seal is located at the bottom of the inlet. It is accessed through the nut in the oven where the column comes out of the inlet. Use ultraclean technique at all times when handling the gold seal. 1. Cool the inlet to 80oC to minimize oxidation and prevent burns. 2. Turn off the carrier gas flow (follow the procedure listed above for Changing the Liner and Septum). 3. Remove the column from the inlet. 4. Remove the insulator cup to reveal the nut housing the gold seal. 5. Remove the nut and turn upside down to remove the gold seal and washer. 6. Insert the new washer and gold seal into the nut: a. Insert the washer into the nut; the washer goes between the gold seal and the nut. b. Insert the gold seal into the nut with the grooves visible on top. (These grooves are the exits for the split gas during split injection and for the purge flow after a splitless injection.) 7. Reinstall the nut containing the gold seal and washer and tighten. 8. Replace the insulator cup over the nut (if present). 9. Replacing the ferrule and trimming the column maybe required (described below). 10. Reinstall the column and check for leaks with a leak detector. (If you turned off the carrier gas, turn it back on and let it flow for a few minutes before you check for leaks.) 11. Turn on the inlet temperature. 12. A blank should be run to bake out the system before analyzing samples. Trimming the Column and Installing a New Column (Inlet/Detector):
Tools needed: column cutting wafer or crystal, inch wrench NOTE: Hydrogen and airflow to the detector must be turned off any time the detector end of the column is removed from the FID unit. The inlet end of the column may be cut several inches to remove active sites and restore separation capacity. 1. 2. 3. 4. 5. 6. Cool the inlet and detector to 80oC. This will minimize oxidation and prevent burns. Turn off the carrier flow following the procedures in Changing the Liner and Septum. Remove the column from the inlet and detector by loosening the column nuts. Remove the column nut and ferrule. The ferrule, which is made of graphite, may be stuck to the nut; remove all ferrule particles. Slide a septum, column nut, and ferrule onto the free ends of the column (tapered end of the ferrule should point away from the column nut, the flat side toward the nut). Cut two to three inches off the end of the column: a. The column should be cut by scoring one side with a wafer or column cutting crystal and then snapping the column at the score. b. Inspect this cut with a magnifying glass. This cut must be clean and contain no rough edges. If there are rough edges, repeat until a clean cut is achieved or seek guidance. c. Wipe the end of the column with a Kimwipe and methanol, or hexane. d. Place the column back into the inlet. When installed, the column should protrude 5 mm (4 mm to 6 mm) beyond the ferrule into the inlet. Use the septum as a guide for this measurement. e. Tighten the column an additional to turn so that the column should not slide with a gentle tug. The detector end of the column should be prepared in the same manner. Position the column so that it extends beyond the end of the nut by manufactures specifications. Use the septum as a guide for this measurement. Tighten the column as described above. Turn on the carrier gas, and check for leaks with a leak detector.
7. 8. 9.
Conditioning the Column: If conditioning is not done properly the column may be ruined. 1. Allow the carrier gas to flow through the column for approximately one hour with the GC oven at room temperature. 2. Ramp the oven temperature at 10-15 degrees per minute to the final conditioning temperature. The final conditioning temperature should be at least 10 degrees higher than the maximum oven temperature to be used in the method but may not exceed 10 degrees below the maximum operating temperature of the column as recommended by the manufacturer. 3. Condition the column several hours or overnight. a. Cool the oven. Cut approximately 2 inches from the detector end of the column as described in Cutting the Column. b. Install the detector end of the column if it has not been done previously.
Dallas County Institute of Forensic Sciences Toxicology Laboratory GC Maintenance 6890 Version 2.0
Cleaning FID Jets or Replacement: Refer to HP 6890 Series Gas Chromatograph Maintenance and Troubleshooting Manual. Cleaning the Split Side Arm: The split vent side arm is the exhaust for split gasses and compounds that are purged off during an injection. This can become very dirty and without maintenance can cause deleterious results. 1. 2. 3. 4. 5. Cool the injector temperature and turn off the gasses. Remove the liner from the inlet as specified in the Changing the Liner and Septum section. Remove the autosampler tray and tower and the top rear instrument cover and fan cover. Remove the split side arm from the inlet to the filter located at the rear of the instrument. Inspect the ends of the side arm to make sure they are not clogged. If they are, use an old syringe to unclog the ends. 6. Using vacuum, pull a solvent such as chloroform through the side arm and into waste. 7. Repeat step 4 using a solvent such as methanol and then room air to dry. 8. Using a small brush or Q-tip dipped in chloroform clean out the inlet arm where the side arm attaches. 9. Reconnect the side arm to the inlet and the filter. 10. Replace the covers and autosampler tray and tower. 11. Change the liner, o-ring and septum. 12. Turn on the temperatures and gases. 13. Check for leaks.
Gas Chromatography/Mass Spectrometry CALIBRATION AND MAINTENANCE Agilent/HP 6890 GC with 5973 Series Mass Spectrometer All chemists are responsible for ensuring the proper operation of gas chromatographs (GC). A chemist is assigned primary responsibility for overseeing maintenance and troubleshooting of the GCs. All chemists are responsible for running QC samples, evaluating instrument calibration, and providing general housekeeping of instrumentation as needed. I. Calibration A. Standard Spectra Tune
The Standard Spectra Tune ensures standard response over the full mass range. This tune is recommended to optimize mass spectral library searches. Therefore, calibration of the GC/MS is performed each day of use by using the Standard Spectra Tune. Tuning should be performed after instrument maintenance and before operation. For the 5973 instruments tune as follows: 1. Select the instrument control screen. 2. Select the Instrument menu item. 3. Select Perform MS Autotune menu item. 4. Select tune to perform Standard Spectra Tune. 5. Review the tune using criteria listed below. Acceptable criteria for the Standard Spectra Tune are as follows: 1. 2. 3. 4. 5. 6. 7. Low background: less than 200 peaks Low water and air: less than 10% Correct mass assignments 0.2 amu (69, 219, 502) Symmetrical, smooth mass peak shapes Consistent mass peak widths (0.6 0.1) Isotope mass assignments should be 1 amu greater than parent peaks Appropriate EM voltage 1000-2800 electron volts. If the voltage is not within these limits, review the history of the electron multiplier or consult a supervisor. The EM voltage will increase over time as the source becomes dirty with use. Cleaning the source should return the EM voltage to a normal operating level. (If it does not, the EM may be going bad.) 8. Mass 69 abundance should be 150,000-350,000 9. Typical relative abundance: - Mass 69 = 100 % - Mass 219 = 35-130% - Mass 502 = 2-5 % 10. Proper isotope ratios:
Dallas County Institute of Forensic Sciences Toxicology Laboratory GC/MS Maintenance 6890/5973 Version 2.0
Mass 70/69 = 0.5-1.6 % Mass 220/219 = 3.2-5.4 % Mass 503/502 = 7.9-12.3 %
In addition, the standard auto tune sets targets for the percent relative abundances for certain other PFTBA masses. The system will come as close as possible to the values shown: - Mass 50 1 % - Mass 131 55 % - Mass 414 3.5 % B. Autotune Autotune maximizes instrument sensitivity over the mass range, using PFTBA masses 69, 219, and 502. This tune is used in troubleshooting instrument operation. A Standard Spectra Tune is performed after the Autotune and prior to routine operation. Acceptable criteria for the Autotune is as follows: Low background: less than 200 peaks Low water and air: less than 10% Correct mass assignments 0.2 amu (69, 219, 502) Symmetrical, smooth mass peak shapes Consistent mass peak widths (0.6 0.1) Isotope mass assignments should be 1 amu greater than parent peaks Appropriate EM voltage: 1000-2800 electron volts. If the voltage is not within these limits, review the history of the electron multiplier or consult a supervisor. The EM voltage will increase over time as the source becomes dirty with use. Cleaning the source should return the EM voltage to a normal operating level. 8. Mass 69 abundance should be 150,000-350,000 9. It is normal at times to have a base peak of 219 instead of 69 10. Relative abundance: - Mass 69 = 100 % - Mass 219 = 35-130% - Mass 502 = 3 % 11. Isotope ratios: - Mass 70/69 = 0.5-1.6 % - Mass 220/219 = 3.2-6.4 % - Mass 503/502 = 7.9-12.3 % C. Quick Tune 1. 2. 3. 4. 5. 6. 7.
Quick Tune provides re-tuning for optimum response and resolution, and for accurate mass assignment. Only the mass axis, peak widths, and EM voltage are adjusted; the lenses are unaffected. This tune may be used to rapidly check tuning after maintenance. The Standard Spectra Tune should be run prior to routine instrument operation.
D.
Failure of a Tune to Meet Acceptable Criteria:
If the Autotune or Standard Spectra Autotune fails, the operating chemist will take note of any error messages generated by the Chemstation Software, check all sources of leaks for tightness, and inform a supervisor. The supervisor will evaluate instrument operation and try to correct the problem. If the problem is corrected, an Autotune and Standard Spectra Autotune shall be performed, and applicable technical, maintenance, and/or repair information documented in the GC/MS Maintenance Logbook. If the problem cannot be resolved, the instrument will be marked as out of service, and the supervisor will arrange for instrument repair. II. Maintenance GC/MS maintenance is documented in the GC/MS Maintenance Logbook located near the instruments. GC/MS Maintenance Schedule Daily/with use Tune: Standard Spectra Autotune Change wash vial solvents Check gases Change liner and o-ring Replace rough pump oil Change gold seal Clean Source Replace septum Trim column Change gas tanks Replace column Change filament Clean split side arm
Bi-Weekly Yearly As needed
Maintenance activities should be consolidated to minimize instrument down time. For example, if it is time to change the gold seal and cut the column, it may be more prudent to change the liner and septum at that time or hold off changing the gold seal until the liner and septum are changed. The maintenance interval is not a rigid schedule and should be based upon workflow. Unless there is an immediate problem, a chromatographic run should continue to completion, and the maintenance performed at the end of a run. Where duplicate instrumentation exists, only one instrument at a time should be scheduled for preventive maintenance to avoid multiple instruments being out of service at the same time.
Ultraclean Technique: Liners, gold seals, and certain other parts must be handled using ultraclean techniques to avoid contamination of the part with oils from the skin, plasticizers from plastic laboratory benchcoats, etc. Do not handle an ultraclean part with your hands; wear cotton gloves or use a fresh Kimwipe. Lay the ultraclean part on its cloth wrapping or on a clean Kimwipe. Changing the Wash Vial Solvent: Discard waste solvent into the hazardous waste. Replace spent solvent in wash vials with fresh methanol, and replace the vials in the appropriate location on the instrument autosampler. Changing the Liner and Septum: Tools needed: inlet wrench and tweezers. NOTE: Turn off carrier gas; if the septum nut is removed without turning off the carrier gas, the glass wool plug may be blown out. Use ultraclean technique at all times when handling the new liner. 1. Cool the inlet to 80oC to minimize oxidation and prevent burns. 2. Turn off the carrier gas on the GC by using the software located on the front panel on the GC: From the front panel on the GC, push the FRONT INLET button. Scroll down using the button to the Total Flow line. Turn off the flow by pressing the OFF button. Once maintenance is complete, turn the flow back on by pressing the ON button. 3. Once the inlet is cool and the gas is off, remove the top septum nut. Remove and replace the septum. Reinstall and tighten the top septum nut. These are not ultraclean parts but care should be taken to avoid any unnecessary contamination. 4. Remove the nut covering the liner. Remove the old liner and discard. 5. Using ultraclean technique, carefully slide an o-ring over the end of the new liner, approximately from the top of the liner, and slide into the inlet. The glasswool plug, if present, should be nearest the bottom of the liner. 6. Replace and tighten the nut and turn on the inlet temperature and carrier gas flow. 7. A blank should be run to bake out the new liner before analyzing samples. Changing the Gold Seal: Tools needed: appropriate screw driver (Phillips/Torx/etc.), 9/16 wrench, wrench Note: The gold seal is located at the bottom of the inlet. It is accessed through the nut in the oven where the column comes out of the inlet. Use ultraclean technique at all times when handling the gold seal.
GC/MS Maintenance 6890/5973 Version 2.0
1. Cool the inlet to 80oC to minimize oxidation and prevent burns. 2. Turn off the carrier gas flow (follow the procedure listed above for Changing the Liner and Septum). 3. Remove the column from the inlet. 4. Remove the insulator cup to reveal the nut housing the gold seal. 5. Remove the nut and turn upside down to remove the gold seal and washer. 6. Insert the new washer and gold seal into the nut: a. Insert the washer into the nut; the washer goes between the gold seal and the nut. b. Insert the gold seal into the nut with the grooves visible on top. (These grooves are the exits for the split gas during split injection and for the purge flow after a splitless injection.) 7. Reinstall the nut containing the gold seal and washer and tighten. 8. Replace the insulator cup over the nut (if present). 9. Replacing the ferrule and trimming the column maybe required (described below). 10. Reinstall the column and check for leaks with a leak detector. (If you turned off the carrier gas, turn it back on and let it flow for a few minutes before you check for leaks.) 11. Turn on the inlet temperature. 12. A blank should be run to bake out the system before analyzing samples. Trimming the Column (Inlet): Tools needed: column cutting wafer or crystal, inch wrench The inlet end of the column may be cut several inches to remove active sites and restore separation capacity. 1. Cool the inlet to 80oC. This will minimize oxidation and prevent burns. 2. Remove the column from the inlet. 3. Remove the column nut and ferrule. The ferrule, which is made of graphite or graphite/vespule, may be stuck to the nut; remove all ferrule particles. 4. Slide a septum, column nut, and ferrule onto the free end of the column (tapered end of the ferrule should point away from the column nut, the flat side toward the nut). 5. Cut two or three inches off the end of the column: a. The column should be cut by scoring one side with a wafer or column cutting crystal and then snapping the column at the score. b. Inspect this cut with a magnifying glass. This cut must be clean and contain no rough edges. If there are rough edges, repeat until a clean cut is achieved or seek guidance. c. Wipe the end of the column with a Kimwipe and methanol, or hexane. d. Place the column back into the inlet. When installed, the column should protrude 5 mm (4 mm to 6 mm) beyond the ferrule into the inlet. Use the septum as a guide for this measurement. e. Tighten the column an additional to turn so that the column should not slide with a gentle tug. 6. Turn on the carrier gas, and check for leaks with a leak detector. Installing a New Column:
Tools needed: column cutting wafer or crystal, inch wrench, MSD installation tool 1. Cool the injector and vent the detector. After the MS has vented, open the vacuum manifold. 2. Turn off the carrier flow following the procedure in Changing the Liner and Septum. 3. Loosen the column nuts from the injector and the transfer nut from the detector, and remove the column. 4. Place a nut and ferrule on each end of the column. (The flat side of the ferrule goes toward the inlet nut and tapered side out, and the cone side slides into the MS transfer line nut, with the flat side out.) NOTE: These are different types of ferrules. See supervisor for instruction. 5. Cut about inch from the inlet end of the column using techniques described in Cutting the Column. 6. Install the new column into the inlet. 7. An MS column, which has been developed for use in a mass selective detector has extremely low column bleed and can be conditioned while in the detector. MS columns can also be conditioned prior to installation into the GC/MSD interface. 8. The column can be installed in the mass spec detector either with an installation tool or without. a. Without the tool: Clean the outside of the column with methanol or hexane, slide the column into the GC/MSD interface until it protrudes 1-2 mm past the end of the interface, hand tighten the nut, and tighten the nut to turn. (Check tightness after one or two vent cycles. b. With the tool: Slide the column into column installation tool, trim 1-2 cm off the end of the column, clean the outside of the column with methanol or hexane, adjust the column so it protrudes 1-2 mm past the end of the tool, hand tighten the nut, slide the septum to touch the end of the nut, use two wrench to tighten the nut turn, remove the column and nut from the installation tool (total length from the septum to the end of the column is 176 mm), clean the outside of the column with methanol or hexane, insert the column into the interface, and tighten the nut to turn. (Check tightness after one or two vent cycles. 9. Close the vacuum chamber, and pump down the mass spectrometer. Conditioning the Column: Tools: Carrier gas, wrench If conditioning is not done properly the column may be ruined. 1. Allow the carrier gas to flow through the column for approximately 5-15 minutes with the GC oven at room temperature 2. Ramp the oven temperature at 5-15 degrees per minute to the final conditioning temperature. The final conditioning temperature should be at least 10 degrees higher than the maximum oven temperature to be used in a method. Do not exceed the manufacturers recommended maximum operating temperature. 3. Hold this temperature and allow carrier gas to flow for several hours, or overnight. 4. Return the GC oven temperature to a low standby temperature.
Cleaning the Split Side Arm: The split vent side arm is the exhaust for split gasses and compounds that are purged off during an injection. This can become very dirty and without maintenance can cause deleterious results. 1. 2. 3. 4. 5. Cool the injector temperature and turn off the gasses. Remove the liner from the inlet as specified in the Changing the Liner and Septum section. Remove the autosampler tray and tower and the top rear instrument cover and fan cover. Remove the split side arm from the inlet to the filter located at the rear of the instrument. Inspect the ends of the side arm to make sure they are not clogged. If they are, use an old syringe to unclog the ends. 6. Using vacuum, pull a solvent such as chloroform through the side arm and into waste. 7. Repeat step 4 using a solvent such as methanol and then room air to dry. 8. Using a small brush or Q-tip dipped in chloroform clean out the inlet arm where the side arm attaches. 9. Reconnect the side arm to the inlet and the filter. 10. Replace the covers and autosampler tray and tower. 11. Change the liner, o-ring and septum. 12. Turn on the temperatures and gasses. 13. Check for leaks. Cleaning the Source for Mass Spectrometers/Changing the Filament/Fill Autocal Vial/ Change Rough Pump Oil: Follow instructions provided in the Agilent/HP 5973 Hardware Manual.
PROCEDURE: ANALYTICAL BALANCE 1) Monthly Calibration a) At the beginning of each month, analytical balances should be calibrated and their linearity checked using certified weights that are located in the Drug Lab. b) Using the Balance Maintenance Log Calibration Check logsheet, record the date, actual weight, and the difference in the last decimal place measured. c) This difference must be within +/- 3 counts in the last place measured. d) If the difference is out of control, then the balance must be evaluated and recalibrated. i) Ensure that the balance is level and clean. ii) Refer to the balance manual for calibration procedure or seek supervisory assistance. e) Once the balance is recalibrated, the linearity should be checked using three certified weights (lower, middle and upper range), and the results recorded on the Balance Maintenance Log - Calibration Check. Also, include the chemists initials, the date, the certified weights used, actual weight measured, and the difference between the last decimal place measured and the target weight. 2) Daily Calibration Check a) Each day before the balance is used, the calibration must be checked. b) On the Balance Maintenance Log sheet record whether the balance is level and clean. c) Also record the difference (in counts) between the measured secondary weight and its target weight. d) The difference must be within +/- 3 counts in the last place measured. e) If the difference is out of control, then the balance must be recalibrated. i) Refer to the balance manual for the calibration procedure or seek supervisory assistance as needed. f) Once the balance is recalibrated, the linearity should be checked using three certified weights (lower, middle and upper range), and the results recorded on the Balance Maintenance Log Calibration Check logsheet. Also, include the chemists initials, the date, certified weights used, actual weights measured, and the difference in the last decimal place measured. 3) For balances with flashing recalibration messages: a) If the secondary weight is within +/- 3 counts after recalibration, no further action is necessary other than recording results in Balance Maintenance Log. 4) For balances with automatic recalibration: a) No additional action is needed other than daily calibration checks. 5) Seek supervisory assistance in any situation in which a balance cannot be calibrated properly or when balance operation appears unusual.
Analytical Balance Version 2.0
PROCEDURE: MEASURING pH More detailed instrument instructions are available in the Accumet Research AR25- Users Manual, located with the instrument and the insert that the electrode was shipped with the VWR International 236123-001 Rev. D. Equipment and Reagents pH Electrode-epoxy with Ag/AgCl internal reference systems and Posi-pHlo junctions Accumet Research AR25 pH meter pH 4 buffer pH 7 buffer pH 10 buffer electrode filling solution electrode storage solution rinsing bottle of deionized water Sample requirements: 1) Aqueous solutions are acceptable; do not use the electrode with organic solvents. 2) Tris, proteins and sulfides can be measured. 3) Proteins will need extra electrode care due to the coating that may form on the bulb. Preparation of the Electrode: 1) Gently remove the black rubber bulb cover and carefully snap the tip protector on if it is not on. 2) Clean any salt deposits from the exterior by rinsing with distilled water. 3) Turn the clear collar so that the fill hole is open. a) The solution should cover the reference junction and be at least one inch above the sample level. b) The fill hole should be open for all measurements. 4) Remove air bubbles 2-Point Standardization: 1) Select two standard buffers which bracket the expected sample pH. 2) Always rinse electrode with deionized water between samples. 3) Touch the standby screen touch anywhere to proceed with channel selection. 4) For the pH electrode choose 1. 5) Then touch pH to define Channel 1 as a pH electrode. 6) Touch std to start standardization process. 7) Touch clear to delete the previous standardization. 8) Place the electrode in the pH 4.0 buffer solution. 9) Wait for stable to appear below the numerical pH reading. 10) Touch std once a stable reading is measured. 11) You will then be prompted to enter the pH value of the buffer. 12) Touch std to standardize with pH 10.0 buffer.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 pH Version 2.0
13) Rinse the electrode with deionized water. 14) Place electrode in pH 10.0 buffer. 15) Repeat steps 8-10 above with the second buffer. 16) When complete the pH values of the buffers will be displayed on the screen. 17) Rinse electrode with deionized water. 18) Check the standardization using the pH 7.0 buffer. 19) Evaluation Criteria a) The value of the check sample must be +/- 0.1 of the actual value. b) The slope should be 96.0% or better. c) Write both of these values on the pH Log Book sheet. Analytical Procedure: 1) 2) 3) 4) Rinse the electrode with deionized water. Place a portion of the sample to analyzed into a clean beaker or test tube. Place the electrode in the sample. Record the pH.
Returning pH Meter to Standby: 1) From the measure screen, touch mode, channel, stdby., 2) Close the fill hole by turning the clear collar and carefully remove the tip protector. 3) Check that the cotton in the black rubber bulb cover is moist. a) If no, add a little pH 4 or pH 7 buffer. 4) Replace the black rubber bulb cover. Electrode Storage: 1) The reference junction must not be allowed to dry out. 2) Do not store in distilled water. a) Storage less than one week between use i) Soak the electrode in pH Electrode Storage Solution (1) A temporary substitute is pH 7 buffer w/ 1g KCl. b) Storage greater than one week between use i) Clean the electrode. ii) Fill electrode with fill solution and cover the fill hole. iii) Cover the sensing surface with the protective cap containing a few drops of storage solution. Electrode Cleaning: 1) Rinse off any salt build-up with distilled water or warm tap water if salt deposits are hard to remove. 2) Drain the reference chamber, and refill with filling solution.
pH Version 2.0
PIPET CALIBRATION Using Pipette Tracker Software Using the Pipette Tracker Software A summary of operation procedures is located at the end of this procedure. Pipette Tracker keeps information about pipettes in device files, which list details for each pipette. This program also uses separate methods, each defining how the calibration should be done. The method includes details on the number of expected volumes to be calibrated for the run, the number of samples to be tested for each expected volume, and the frequency of evaporation blanks. The method also specifies the testing criteria to be used in determining whether the calibration passes or fails. When a devices interval determines that it is due for a calibration, the device will appear automatically on the built-in WorkList. A time (reminder) has been specified for each interval, which will place the device on the WorkList before it is due to allow you to plan you your calibrations. When calibrating, the weights will be automatically collected from the balance and put into the computer. You simply follow the on-screen steps and the Pipette Tracker performs all the calculations, providing instant feedback if the device passes or fails the calibration and generates a report. This report will be kept by the laboratory Quality Manager. Logon/Logoff The program will ask for the users password each time the program is started. Each user must log on by entering his or her password in the [Logon] dialog box so that the program can record it. Once the log on has taken place, the program will automatically insert the users name into the operator field on the Calibration Run screen. The user can log off by pulling down the Password menu and select Logoff. Only certain components of the program will remain active. The next user may then log on by pulling down the Password menu and selecting Logon. Run Calibration There are three ways to get to the Run Calibration screen: From the WorkList, you can select a device and pull down Device menu and select Run Calibration. The currently highlighted device and method will be loaded. From the Inventory List, you can select a device and pull down Device menu and select Run Calibration. The currently highlighted device and the most due method (or the closest to being due) will be loaded. From the Device Setup screen, you can click on the Cal button to calibrate that particular device. The currently edited device and the most due method (or the closest to being due) will be loaded. Calibration Run Screen
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Pipette Calibration Version 2.0
The top entries and the Environmental factors (Temp., Barom. P., Rel. Hum.) can be edited before Starting a Run. Once started, you are locked out of these areas. After the last weight is received, the Environmental factors can be revised again, if necessary. If these are changed after the last weight is received, the program will also recalculate the calibration results and statistics. After confirming the correct Input Mode (Balance), shown in the Balance ID field, ensure that the device and method are the ones you want before running the calibration. The following describes some of the components (others found in the manual) of the Run Calibration screen: Temp. -- This field contains the liquids temperature in degrees Centigrade. This information can be read from the meter found on the wall behind the balance. As noted earlier, this can only be edited at the start of a Calibration Run. Barom. P. -- This field contains the barometric pressure in mm of mercury ( inner most scale times 10). This information can be read from the barometer found on the wall behind the balance. As noted earlier, this can only be edited at the start of a Calibration Run. Rel. Hum. -- This field contains the value for the relative humidity, with the units as %. This information can be read from meter found on the wall behind the balance. As noted earlier, this can only be edited at the start of a Calibration Run. Abort Run -- Deletes all current weight, time and volume information for this run. Environmental factors are not changed. Device selection, operator name and method are not changed. Redo Sample -- This button becomes active once you have entered the Tare weight and the first calibration sample. Clicking on it cancels the results entered for the most recent sample only, and resets the Run Line to the previous sample so you can re-send the weight from the balance. Please note the Redo Sample button does not allow you to re-dispense the sample, only to correct an error in entry. If you have a problem with dispensing the sample, consider using the Void button at the end of the Run (see details below), or abort the calibration and start again. Void Sample -- This button becomes active only at the end of the Calibration Run, and before you click on Accept Run. If you experience problems dispensing one or more samples, you may choose to exclude these from the calculations for the Run, rather than abort the Run and start over. In effect, the calculations are performed with fewer sample(s). When the Run is finished, you will be able to move the cursor up and down through the cumulative weights shown on the Calibration Run display grid. Position the cursor over the sample to be voided, then click on the Void button. The calculated Volume for this sample will be replaced with the word VOID. You are able to Unvoid the sample by positioning your cursor over the voided cumulative weight and clicking on the Void button again. This reverses all effects of clicking Void. Accept Run -- This button only becomes active when the run is complete. Clicking on it accepts the Calibration Run, stores the results, then inactivates the button and the report will be automatically printed. Done -- Leaves the Calibration Run screen and goes back to the WorkList or Inventory List.
Pipette Calibration Version 2.0
Run Calibration Automatically (Through the Balance) First, ensure that the balance is calibrated properly by following the QA Procedure for the Analytical Balance, then proceed as follows from the Run Calibration screen: Making sure that the correct device (pipette) and method are listed, edit the Environment factors (Temp., Barom. P., Rel. Hum.) Start the calibration by clicking on Start Run button. Place your container with liquid on the balance. Tare the balance and click on Tare Wgt, which sends the weight over to the Calibration Run Table. Important: Tab over to Sample Wgt. Pipette a volume into the container. Once a stable weight has been obtained, press Enter, which sends the weight over to the Calibration Run Table. Continue until all volumes have been recorded. Using Abort, Void Sample and Redo as necessary. At the completion of the calibration , either click on Accept Run or Abort Run. Print a hard copy of the report, sign and file next to the balance or with the Quality Manager. Another device may be chosen by pulling down the device list. Once the calibration(s) are completed, click on Done. Log off by clicking on Password and choosing LogOff.
Calibration of Pipetters
Procedure Summary
There may be three reasons for checking calibration of a pipetter: Receiving a new pipetter into the laboratory. See the Quality Manager prior to calibration. Regularly scheduled calibration. Suspected error in calibration. A condensed summary follows: The computer, printer and balance should be on. If not, allow the balance to warm up for thirty minutes. With the 20 gram weight, check the balance calibration and record weight on the Balance Calibration Log. See Quality Manager if balance is not in calibration. Log on to the computer by pulling down the Password menu and selecting Logon. If you do not have a password, see Quality Manager. From the WorkList, select the pipetter that is to be calibrated. Pull down the Device menu and select Run Calibration. From the meters on the wall, enter the current temperature, barometric pressure and relative humidity. Begin the calibration by clicking on Start Run button. Place container with enough liquid to cover the bottom on to the balance. Tare the balance and click on the Tare Wgt button, which sends weight over to the computer Calibration Run Table. Important: Tab over to Sample Wgt button. Pipette a volume into the container. Once a stable weight has been obtained, press Enter, which sends the weight over to the computer Calibration Run Table. Continue until all volumes have been recorded. Using AbortRun, VoidSample and Redo as necessary. At the completion of the calibration, either click on Accept Run or Abort Run. Print a hard copy of the report, sign and file next to the balance or with the Quality Manager. Another pipetter may be chosen by pulling down the device list. Once the calibration(s) are completed, Click on the Done button. Log off the computer by pulling do down the Password menu and selecting Logoff. If pipetter performance is acceptable, the printed report is filed next to the balance or given to Quality Manager. If pipetter performance is not acceptable, immediately remove from service and notify Quality Manager.
REAGENT LOGBOOK ALCOHOLS & ACETONE

Critical Reagents ---Stocks, Standards, Controls, Calibrators: 0.012% n-Propanol (internal standard solution): Transfer 0.72 grams of n-propanol to a 6 liter volumetric flask and dilute to volume with distilled water. 0.015% External Ethanol Control: Pre-made NIST traceable standard. 0.08% External Ethanol Control: Pre-made NIST traceable standard. 0.20% External Ethanol Control: Pre-made NIST traceable standard. 0.05% External Multi-component Alcohol Mix Control: Pre-made NIST traceable standard. This mix contains ethanol, methanol, isopropanol, and acetone. 0.10% External Multi-component Alcohol Mix Control: Pre-made NIST traceable standard. This mix contains ethanol, methanol, isopropanol, and acetone. High Mixed Calibration Standard (0.5% ethanol, 0.5% methanol, 0.125% acetone, 0.125% isopropanol): Transfer 0.125 grams each of acetone and isopropanol and 0.50 grams each of ethanol, and methanol to 100 mL volumetric flask and dilute to volume with deionized water. Alternatively a commercially available NIST traceable standard of this or different concentration may be used. Low Mixed Calibration Standard (0.05% ethanol, 0.05% methanol, 0.0125% acetone, 0.0125% isopropanol): Dilute High Mixed Calibration Standard 1:10. For example, transfer 5 ml of the High Mixed Calibration Standard to 50 ml volumetric flask and dilute to volume with deionized water. 0.50% Ethanol Calibration Standard: Transfer 0.50 grams of ethanol to 100mL volumetric flask and dilute to volume with deionized water. Alternatively a commercially available NIST traceable standard of this or different concentration may be used. 0.05% Ethanol Calibration Standard: Dilute 0.50% Ethanol Calibration Standard 1:10. For example, transfer 5 ml 0.50% Ethanol Calibration Standard to 50 ml volumetric flask and dilute to volume with deionized water.
ELISA- UNIVERSAL
Negative Controls consist of blank blood, DI water, or blank urine. Blank blood and Urine are either bought or tested prior to lab implementation. Discrepancies are always reported to a supervisor for evaluation before use. They are NOT to be recorded in the Reagent Logbook. Critical Reagents ---Stocks, Standards, Controls, Calibrators:
I. Sexual Assault ELISA A. Barbiturate (BARB) Controls: 1. Stock - 1 mg/mL secobarbital stock solution in MeOH. a) NOTE: This standard is also used in the Acid Neutral Screen; refer to that
procedure for preparation instructions.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Reagent Logbook Instructions Version 2.0
2. Working Standard 10 ug/mL a) Add 100 uL of 1 mg/mL secobarbital stock to a 10 mL volumetric and bring to
volume with methanol (MeOH).
B. Flunitrazepam Metabolite (FLUZ) Controls: 1. Stock - 1 mL ampule of 100 ug/mL 7-amino flunitrazepam 2. Working Standard 1 ug/mL a) Add 100 uL of 100 ug/mL 7-amino flunitrazepam stock to a 10 mL volumetric
flask and bring to volume with methanol. C. BARB and FLUZ Negative Control (NC): 1. Use approximately 500 uL to 1mL of blank urine. BARB and FLUZ Notes: 1. The BARB (300ng/mL) and FLUZ (5 ng/mL) LC can be prepared in the same test tube by mixing 965 uL of blank urine, 30 uL BARB working standard, and 5 uL FLUZ working standard. 2. The BARB (1500 ng/mL) and FLUZ (25 ng/mL) PC can be prepared in the same test tube by mixing 825 uL of blank urine, 150 uL BARB working standard, and 25 uL of the FLUZ working standard. ** Expiration of the LCs and PCs is 2 weeks from preparation date.
II. THC/COC/OPI ELISA A. Opiate Controls (OPI): 1. Stock - 1mL ampule of 1 mg/mL morphine 2. Working Standard 1 ug/mL a) Add 10 uL of 1 mg/mL morphine stock to a 10 mL volumetric flask and bring
to volume with methanol.
B. THC Metabolite (THC) Controls 1. Stock - 1mL ampule of 100 ug/mL N-nor-9-carboxy-delta-9-THC 2. Working Standard 1 ug/mL a) Add 100 uL of 100ug/mL carboxy-THC to a 10 mL volumetric flask and bring
to volume with methanol.
C. Cocaine Metabolite (COC) Controls: 1. Stock -1 mL ampule of 1 mg/mL benzoylecgonine 2. Working Standard 10 ug/mL a) Add 100 uL of 1 mg/mL benzoylecgonine stock to a 10 mL volumetric flask
and bring to volume with acetonitrile.
D. OPI, THC, and COC Negative Control (NC): 1. Use approximately 500 ul to 1mL of blank blood. E. OPI, THC, and COC Notes 1. Low Control - The OPI (25 ng/mL), COC (100 ng/mL), and THC (20 ng/mL) LC can
be prepared in the same test tube by mixing 945 uL of blank blood, 25 uL OPI working standard, 20 uL THC working standard, and 10 uL of COC 2. Positive Control - The OPI (100 ng/mL), COC (500 ng/mL), and THC (50 ng/mL) PC can be prepared in the same test tube by mixing 800 uL of blank blood, 100 uL OPI working solution, 50 uL THC working solution, and 50 uL COC working solution. 3. Expiration of OPI, THC, and COC LCs and PCs is 2 weeks from preparation date.
Reagent Logbook Instructions Version 2.0
III. LSD ELISA

A. LSD Controls: 1. LSD Stock - 2 mL bottle of 1 ng/mL LSD sent with ELISA kit. 2. LSD LC (0.2 ng/nL) - In at test tube, mix 800ul blank urine and 200 uL of 1 ng/mL LSD stock. 3. LSD PC (1 ng/mL) - Use 0.5 mL of the LSD stock. 4. Expiration of OPI, THC, and COC LCs and PCs is 2 weeks from preparation date. IV. METH ELISA B. Methamphetamine (Meth) Controls: 1. METH Stock 50 ng/mL a) Option 1 Use the 2 mL bottle of 50 ng/mL d-methamphetamine sent with ELISA kit b) Option 2 (1) Prepare a 1mg/mL d-methamphetamine in methanol solution. (a) NOTE: This standard is also used as an ALK response factor; refer to that procedure for preparation instructions. (2) Stock (50ng/mL solution) - In a 100 mL volumetric use 5 uL of the 1mg/mL d-methamphetamine in methanol solution and dilute to volume with blank urine or deionized water 2. METH LC (20 ng/mL) a) Mix 600ul blank urine or deionized water with 400 uL of 50 ng/mL d-meth stock with for the d-meth low control (LC). 3. METH PC (50 ng/mL) a) Use 0.5mL of the ELISA METH stock. 4. Expiration of OPI, THC, and COC LCs and PCs is 2 weeks from preparation date.
ALKALINE DRUG SCREEN

1.0N Hydrochloric acid: Add approximately 500 mL deionized water to a 1 liter volumetric flask, carefully add 83 mL of concentrated hydrochloric acid. Mix, allow to cool as necessary, and dilute to volume with deionized water Critical Reagents ---Stocks, Standards, Controls, Calibrators: 1.0 mg/mL Cholestane Stock Internal Standard: Transfer 50 mg cholestane to a 50 mL volumetric flask and dilute to volume with chloroform. 0.02 mg/mL Cholestane Working Internal Standard: Transfer 2.0 mL cholestane stock internal standard (1.0 mg/mL) to a 100 mL volumetric flask and dilute to volume with chloroform. 1.0 mg/mL Alphaprodine Stock Internal Standard: Add 10 mL deionized water to preweighed alphaprodine free base. 0.1 mg/mL Alphaprodine Working Internal Standard: Transfer 10.0 mL alphaprodine stock standard (1.0 mg/mL) to a 100 mL volumetric flask and dilute to volume with deionized water. 0.06 mg/mL Alkaline QC Mixture: Transfer 3.0 mL of each of the following 1.0 mg/mL stock standards methamphetamine, cocaine, lidocaine, citalopram, and trazodone - to a 50 mL volumetric flask and dilute to volume with methanol.
ALK Retention Index

Dallas County Institute of Forensic Sciences Toxicology Laboratory 3 Reagent Logbook Instructions Version 2.0
1. 2.
In a 15 mL extraction tube, place 4 mL 1N HCl. Add 100 ul of each of the following drug stock standards (1mg/mL) in methanol: amphetamine zolpidem phendimetrazine alprazolam chlorpheniramine verapamil desipramine mesoridazine demethyldiazepam Add 1.5 mL alphaprodine working internal standard solution in water (0.2 mg/L) Add 2 mL chloroform. Add 800uL ammonium hydroxide and cap the tube. Vortex for 60 sec; invert and shake at 15 second intervals during the vortexing period. Centrifuge. Transfer chloroform and some aqueous phase to a 5 mL conical test tube. Transfer a small amount (about 50 uL) of the organic layer to an autosampler vial and inject on both instruments. Remaining solution may be stored for future checks.
3. 4. 5. 6. 7. 8. 9.
ACID NEUTRAL DRUG SCREEN

Toluene-ether (1:1): In a hood, combine equal volumes of toluene and diethylether. Potassium dihydrogen phosphate, saturated: Add 270 grams potassium dihydrogen phosphate to 900 mL water in a beaker containing a stirring bar. Gently warm the solution on a hot plate while stirring. The potassium dihydrogen phosphate will slowly go into solution. Cool before use. Critical Reagents ---Stocks, Standards, Controls, Calibrators: 0.2 mg/mL Barbital Internal Standard: Transfer 50 mg barbital free acid to 250 mL volumetric flask and dilute to volume with deionized water. Make fresh every 6 months A/N Working Standard Mix 0.2 mg/ml (200mg/L): Using a volumetric pipette, transfer 2.0 mL each of a 1.0 mg/ml stock solution of up to 5 standards to a 15 mL screw cap culture tube. Add methanol if needed to bring the total volume to 10 mL. During normal use, this solution is used-up before stability becomes a problem. A/N Working Mix 1: butalbital, meprobamate, carisoprodol, phenobarbital, and phenytoin A/N Working Mix 2: pentobarbital, secobarbital, and primidone A/N QC Mixture 0.1 mg/ml (100mg/L): Using a volumetric pipette, transfer 1.0 mL each of a 1.0 mg/ml stock solution of selected standards to a 10 mL volumetric flask and dilute to volume with methanol. During normal use, this solution is used-up before stability becomes a problem. A/N QC Mix 1: butalbital, meprobamate, carisoprodol, phenobarbital, and phentoin A/N QC Mix 2: pentobarbital, secobarbital, primidone External Control: Biorad Liquichek #2 (has phenobarbital, phenytoin, primidone)
METHOCARBAMOL
Toluene-ether (1:1): In a hood, combine equal volumes of toluene and diethylether. Potassium dihydrogen phosphate, saturated:
Add 270 grams potassium dihydrogen phosphate to 900 mL water in a beaker containing a stirring bar. Warm on a hot plate while stirring until the potassium dihydrogen phosphate
goes into solution. Cool before use.
Critical Reagents: Stocks, Standards, Controls, Calibrators: 0.2 mg/mL Barbital Internal Standard: Transfer 50 mg barbital free acid to 250 mL volumetric flask and dilute to volume with deionized water. Make fresh every 6 months. 1.0 mg/ml Drug Stock Standard: Transfer 10 mg free drug to a 10 mL volumetric flask and dilute to volume with methanol. If the drug is in the form of a salt or contains waters of hydration, the 10 mg must be adjusted accordingly to add 10 mg of the free base. The calculation to convert an amount of free acid to the salt is as follows: mg of salt = mg of free drug x molecular weight of salt / molecular weight free drug Methocarbamol Working Standard 0.2 mg/ml (200mg/L): Using a volumetric pipette, transfer 2.0 mL of a 1.0 mg/ml stock solution to a 10 mL volumetric flask and dilute to volume with methanol. Methocarbamol QC 0.1 mg/ml (100mg/L): Using a volumetric pipette, transfer 1.0 mL of a 1.0 mg/ml stock to a 10 mL volumetric flask and dilute to volume with methanol.
VALPROIC ACID
3N Hydrochloric Acid: Fill a 500 ml volumetric flask approximately half full with deionized water. Add 125 ml concentrated hydrochloric acid. Mix carefully and allow to cool if necessary. Fill to the mark with deionized water Critical Reagents ---Stocks, Standards, Controls, Calibrators: 1.0 mg/ml Thymol Internal Standard: Weigh 10 mg thymol and transfer to a 10 ml volumetric flask. Dilute to volume with methanol. 10 mg/ml Valproic Acid Stock Standard: Weigh 116 mg Valproic acid (sodium salt) and transfer to a 10 ml volumetric flask. Dilute to volume with methanol. External Control: Biorad Liquichek #2
ETHOSUXIMIDE
3N Hydrochloric Acid: Fill a 500 ml volumetric flask approximately half full with deionized water. Add 125 ml concentrated hydrochloric acid. Mix carefully and allow to cool if necessary. Fill to the mark with deionized water.
Stocks, Standards, Controls, Calibrators:

10 mg/ml Ethosuximide Stock Standard: Weigh 100 mg ethosuximide and transfer to a 10 ml volumetric flask. Dilute to volume with methanol.
ETHCHLORVYNOL
3N Hydrochloric Acid: Fill a 500 ml volumetric flask approximately half full with deionized water. Add 125 ml concentrated hydrochloric acid. Mix carefully and allow to cool if necessary. Fill to the mark with deionized water. Critical Reagents ---Stocks, Standards, Controls, Calibrators: 10 mg/ml Ethchlorvynol Stock Standard (such as Alltech cat. 01733, 100 uL vials): Weigh 100 mg ethchlorvynol (will take two vials) and transfer to a 10 ml volumetric flask. Dilute to volume with methanol. Store in light protected tube and below room temperature
ETHYLENE GLYCOL
1% n-Butylboronic Acid (derivatizing agent): Place 50 mg of n-butylboronic acid into a 5 mL volumetric flask. Add 10-15 drops of acetone and mix. Gradually bring to volume with ethyl acetate. Critical Reagents ---Stocks, Standards, Controls, Calibrators: Ethylene Glycol Working Standard (22.26 mg/mL): Place 200 uL of ethylene glycol into a 10 mL volumetric flask and bring to volume with deionized water. (Density of ethylene glycol at 20 deg C = 1.1135 g/mL.) 1,2-Butanediol Working Internal Standard (20.12mg/mL): Place 20 uL of 1,2-butanediol into a 10 mL volumetric flask and bring to volume with deionized water. (Density at 20 deg C = 1.0060 g/mL.)
ACETAMINOPHEN
Potassium dihydrogen phosphate buffer (KH2PO4), saturated: Add 270 grams potassium dihydrogen phosphate to 900 mL water in a beaker containing a stirring bar. Warm on a hot plate while stirring until the potassium dihydrogen phosphate goes into solution. Cool before use. Critical Reagents ---Stocks, Standards, Controls, Calibrators: 0.2 mg/mL Barbital Working Internal Standard: Transfer 50 mg barbital free acid to 250 mL volumetric flask and dilute to volume with deionized water. Make fresh every 6 months 1.0 mg/mL Acetaminophen Working Standard: Transfer 10 mg acetaminophen to a 10 mL volumetric flask. Bring to volume with deionized water. Store in refrigerator. Before using, allow standard to adjust to room temperature.
SALICYLATES
Toluene-Ether (1:1): Under a vent hood, combine 2 liters toluene and 2 liters diethylether. 3 N Hydrochloric Acid: Add approximately 500mL deionized water to a one liter volumetric flask. Add 249mL of concentrated hydrochloric acid. Mix and allow to cool. Dilute to volume with deionized water. 5% Borate Buffer: Transfer 10.0 grams sodium borate to a 200 mL volumetric flask and dilute to volume with deionized water Critical Reagents ---Stocks, Standards, Controls, Calibrators: 200 mg/L Salicylic Acid QC Sample:
Dallas County Institute of Forensic Sciences Toxicology Laboratory Reagent Logbook Instructions Version 2.0
1.0 mg/mL salicylic acid stock standard make the above listed stock standard using salicylic acid standard from another vendor or lot as applicable. Transfer 2.0 mL of 1.0 mg/mL QC salicylic acid stock to a 10 mL volumetric flask and dilute to volume with deionized water.
COCAINE & METABOLITES

Phosphate Buffer, 0.1M, pH 6.0: Dissolve 13.6 g KH2PO4 in 800 ml deionized H20. Adjust to pH 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 with deionized H20. Methylene Chloride/Isopropanol/Ammonium Hydroxide (78/20/2): To prepare 200 mL, combine 4 mL ammonium hydroxide, 156 mL methylene chloride, and 40 mL isopropanol. Prepare fresh daily. Mix reagents in the order listed. Hydrochloric Acid, 0.1M: To 400 ml deionized H20 add 4.2 ml concentrated HCl. Dilute to 500 ml with deionized H20. Mix. Critical Reagents ---Stocks, Standards, Controls, Calibrators: Stock Standards (1.0 mg/ml): Cocaine (COC), Cocaethylene (CE), Ecgonine methyl ester (EME) and Benzoylecgonine (BE) standards. Prepared by manufacturer such as Cerilliant. Corresponding deuterated standards (100 ug/ml): Prepared by manufacturer such as Cerilliant. Cocaine D3 Cocaethylene D3 Ecgonine Methyl Ester D3 Benzoylecgonine D3 Working Internal Standard Mixture (10 ug/ml): Transfer 1.00 ml of each deuterated standard to a 10 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: Same as Working Standard Mixture (prepared independently). External Control: Biorad S2
OPIATES GC/MS
Carbonate Mix (8:3): Mix 80 grams of sodium bicarbonate and 30 grams sodium carbonate and pulverize with a mortar and pestle. Store at room temperature. 0.2N Hydrochloric acid: Add approximately 500 ml deionized water to a one liter volumetric flask. Add 16.6 ml concentrated hydrochloric acid. Mix and allow to cool. Dilute to volume with deionized water. Store at room temperature.
Ethyl acetate/isobutanol (9:1): Add 900 ml of ethyl acetate to a one liter flask. Add 100 ml isobutanol. Mix and store at room temperature. Critical Reagents ---Stocks, Standards, Controls, Calibrators: D3-Stock Internal Standards (100ug/ml each of deuterated morphine, 6-monoacetylmorphine, codeine, hydromorphone, and hydrocodone): Prepared by manufacturer. Store in freezer. D3-Working Internal Standard Mixture (4ug/ml): Using a volumetric pipet, transfer 1.0 ml of each of the following Stock Internal Standards to a 25 mL volumetric flask and dilute to volume with acetonitrile; store in refrigerator: codeine D3 (Stock: 100 ug/ml in methanol) hydrocodone D3 (Stock: 100 ug/ml in methanol) hydromorphone D3 (Stock: 100 ug/ml in methanol) morphine D3 (Stock: 100 ug/ml in methanol) 6-monoacetylmorphine D3 (Stock: 100 ug/ml in acetonitrile) Drug Stock Standard (1.0 mg/ml): Prepared by manufacturer. Store in refrigerator. Drug Working Mix Standard (10 ug/ml): Using a 50 ul Eppendorf pipet, transfer 50 ul each of the following drug stock standards (1.0 mg/ml) to a 5.0 ml volumetric flask and dilute to volume with acetonitrile; store in refrigerator: codeine morphine 6-monoacetylmorphine hydromorphone hydrococone Drug external QC standard (10 ug/ml): Using a 50 ul Eppendorf pipet, transfer 50 ul each of the codeine, morphine, 6-monoacetylmorphine, hydromorphone, and hydrocodone stock standards (1.0 mg/ml) to a 5.0 ml volumetric flask and dilute to volume with acetonitrile. (The vials containing the drug standards used as a check samples are located in the cooler and are labeled Alltech Drug Opiate Stds). Store in refrigerator.
GHB
Potassium phosphate buffer, 0.1 M, pH 6.0: Dissolve 13.6 g KH2PO4 in 800 ml deionized water. Adjust to pH 6.0 by addition of 40% NaOH. Dilute to 1000 ml using deionized water. Mix. Sodium hydroxide, 40%: Dissolve 40g NaOH in deionized water. Cool. Dilute to 100mL with deionized water. Methanol/Ammonium hydroxide solution (99/1): To a 100 ml graduated cylinder add 1 ml of concentrated ammonium hydroxide, and fill to 100 ml with methanol. Prepare fresh daily. Critical Reagents ---Stocks, Standards, Controls, Calibrators: GHB working standard (200 mg/L): Transfer 2.0 ml of GHB to a 10 ml volumetric flask. Dilute to volume with methanol. Standards are ordered from Cerilliant. GHB quality control (200 mg/L): Same as working standard (prepared independently). Standards are ordered from Cerilliant.
GHB deuterated internal standard (100 ug/ml): Prepared by manufacturer. Standards are ordered from Cerilliant.
BENZODIAZPINES
40% Sodium Hydroxide: Dissolve 40g NaOH in approximately 80 mL of deionized water. Allow to cool. Bring to 100 mL total volume with deionized water. pH 11, 0.5M Phospate Buffer: Dissolve 8.7 g K2HPO4 (potassium phosphate dibasic) in approximately 80 mL deionized water. Adjust pH to 11 with 40% NaOH. Bring volume to 100 mL with deionized water. Critical Reagents ---Stocks, Standards, Controls, Calibrators: Oxazepam-d5 Stock Internal Standard (100 ug/mL): Prepared by manufacturer. Store in freezer. Alprazolam- d5 Stock Internal Standard (100 ug/mL): Prepared by manufacturer. Store in freezer. Working d5 Internal Standard (10 ug/mL): Using an appropriate pipet or syringe, transfer 1.0 mL of each of the above listed d5 benzodiazepines to a 10.0 ml volumetric flask and dilute to volume with methanol. Store in refrigerator. Drug Stock Standards: Prepared by manufacturer. Store in refrigerator: Mix 1: Oxazepam 1.0mg/mL Temazepam 1.0mg/mL Lorazepam 1.0mg/mL Alprazolam 1.0mg/mL -Hydroxyalprazolam 0.1mg/mL Mix 2: Flunitrazepam 1.0mg/mL 7-Aminoflunitrazepam 0.1mg/mL Clonazepam 1.0mg/mL 7-Aminoclonazepam 0.1mg/mL Triazolam 1.0mg/mL -Hydroxytriazolam 0.1mg/mL
Mixed Drug Standards (10 ug/mL): Two mixed drug standard solutions are made: Drug Mix 1 and Drug Mix 2 containing drugs as noted above. Into a 5.0 mL volumetric flask labeled Mix 1 or Mix 2 as appropriate, place 50uL of each appropriate 1.0 mg/mL stock drug standard using a 50 uL Eppendorf pipette and500 uL of each appropriate 0.1 mg/mL drug standard Dilute to volume with deionized water. Store in the refrigerator External Control: Biorad
CARBOXY-TETRAHYDROCANNABINOL IN URINE BY GC/MS

10N 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 at room temperature. Make fresh each assay.
Critical Reagents ---Stocks, Standards, Controls, Calibrators: 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 calibrators and 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-D3 Stock Internal Standard (100 ug/ml): Prepared by manufacturer. THC-D3 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 50ul COOHTHC-D3 Stock Internal Standard to a 10 ml volumetric flask and dilute to volume with methanol. Store in the freezer; light sensitive. External QC Sample: Prepared by manufacturer such as Biorad S2.
CANNABINOIDS BY GC/MS
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 ml of 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. Critical Reagents ---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 calibrators and 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. 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 S2.
10
LEAD
2% HNO3 (Make-up solution): In a 200 mL volumetric flask, add approximately 75 mL deionized water and 4 mL of concentrated nitric acid. Mix carefully and bring to volume with deionized water. 10% Triton X-100 (v/v): In a 100 mL volumetric flask place 10 ml Triton X-100 into approximately 70 mL deionized water. Stir for ~ 1 hour. The solution may need to be slightly warmed or sonicated for complete dissolution. Bring to volume with deionized water.. Expires One month after preparation date. 20% NH4H2PO4 (w/v): In a 100 mL volumetric flask, dissolve 20g of NH4H2PO4 in ~75 mL deionized water. Bring to volume with deionized water. Expires Six months from prep date. Critical Reagents ---Stocks, Standards, Controls, Calibrators: Lead Working Modifier: In a 100 mL volumetric, add ~ 30 mL of deionized water, 5 mL of 10% Triton X100, 1 mL of 20 % NH4H2PO4, and 200 L concentrated nitric acid. Mix. Bring to volume with deionized water. Expires 3 weeks from prep date. 10g/mL Intermediate Pb Standard: Into a 100 mL volumetric, pipet 1.0 mL stock Pb standard (1000 ug/mL purchased standard) and 2.0 mL concentrated nitric acid into ~75mL deionized water. Mix. Bring to volume with deionized water. Expires 1 week from prep date 400 g/L Pb High Calibration Std*( PbHC Std A )*: Into a 50 mL volumetric flask, pipet 2 mL of 10ppm intermediate Pb Standard and bring to volume with 2% HNO3. Expires - Make fresh daily (24 hr expiration) 200 g/L Pb High Calibration Std*( PbHC Std B )*: Into a 50 mL volumetric flask, pipet 1mL of 10ppm Intermediate Pb Standard and bring to volume with 2% HNO3. Expires - Make fresh daily (24 hr expiration) Positive Control (300 ug/L) : Prepare in 2mL auto sample cup. Add 900L of Lead Working Modifier and 75L of PbHC std A + 25L 2% HNO3 Expires - Make fresh daily (24 hr expiration)
CYANIDE
0.10 N Sodium Hydroxide: Transfer 4.0 grams sodium hydroxide to a one liter volumetric flask and dilute 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 and then add 167 mL concentrated sulfuric acid. Mix and allow to cool. Dilute to volume with deionized water. Store at room temperature. Saturated Lead Acetate: Transfer 1.0 g lead acetate to a 10 mL volumetric flask and dilute to volume with 1.0N sodium hydroxide. Store at room temperature.
11
Critical Reagents ---Stocks, Standards, Controls, Calibrators: Potassium Cyanide Stock Standard (1000 ug/mL): Transfer 250 mg of potassium cyanide to a 100 mL volumetric flask and dilute to volume with 0.1N sodium hydroxide. Stable for 6 months at 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 for the calibrators and one for the quality control. Using an Eppendorf pipet, transfer 1.0 mL cyanide stock standard (1000 ug/mL) to a 10 mL volumetric flask and dilute to volume with 0.1N sodium hydroxide. Stable for one day only.
ARSENIC
6N HCl: Fill a 200 ml volumetric flask with about 75 ml deionized water. Add 100mL of concentrated HCl and swirl. Allow to cool and bring to volume with deionized water. Nitric : Sulfuric Acid (50:50) (digestion acid): In a hood using eye protection, place 50mL of concentrated nitric acid into a glass stoppered container. Add 50mL of concentrated sulfuric acid. Carefully swirl for good mixing of the two acids. Allow to cool before use. 20% Urea (w/v): Place 20g of urea into a 100mL volumetric flask. Bring to volume with deionized water. Endothermic Reaction Make fresh for each set. 20% Potassium Iodide (w/v): Place 10g of KI into a 50mL volumetric flask. Bring to volume with deionized water. Endothermic Reaction Make fresh for each set. Critical Reagents ---Stocks, Standards, Controls, Calibrators: 0.5 % NaOH / 0.6 % NaBH4 (Reductant): Place 0.5g NaOH and 0.6g NaBH4 in a 100 mL volumetric flask and bring to volume with deionized water. Make fresh for each set. 2500 g/L Intermediate Arsenic Standard A: Into a 50 mL volumetric, pipet 125L Stock Arsenic Standard (1000 ug/mL purchased standard). Dilute to volume with deionized H20. Expires 1 week from prep date. Positive Control (50ug/L): Add 2.5mL of blank blood to a 50mL digestion tube, spike with 50L of Arsenic Standard A swirl to mix. External Positive Control -Urine Metal Control Level 1: Add 2.5mL of Urine Metal Control Level 1 or 2 to a 50mL digestion tube
LITHIUM
4000 g/mL KCl: In a 200 mL volumetric flask, place 0.8g of KCl and bring to volume with deionized water. Critical Reagents ---Stocks, Standards, Controls, Calibrators:
12
100g/mL Intermediate Li Standard A: Into a 10 mL volumetric, pipet 1mL stock lithium standard (1000 ug/mL purchased standard). Bring to volume with deionized H20. Expires 1 week from prep date Positive Control (2.0mEq/L): Add 1mL of blank blood to 4mL of deionized water + 5mL of KCl + 140L of Li Standard A in a 25mL test tube. Invert gently to mix. Expires - Make fresh daily (24 hr expiration) External Control: Add 1mL of BioRad Level 2 and 4mL of deionized water + 5mL of KCl in a 25mL test tube. Invert gently to mix. Expires - Make fresh daily (24 hr expiration)
MERCURY
5:2:1 Nitric : Perchloric: Sulfuric Acid (digestion acid): In a hood with eye protection, place 500mL of concentrated nitric into a glass-stoppered container. Add 200mL of concentrated perchloric acid. Add 100mL of concentrated nitric acid. Carefully swirl to mix. Allow to cool before use Critical Reagents ---Stocks, Standards, Controls, Calibrators: 25% Stannous Chloride (reductant): In a hood, place 25g Sn2Cl, 25mL of deionized H20, and 20 mL of HCl into a 100 mL volumetric flask. Swirl. Bring to volume with deionized H20. Expires Make fresh for each set. 5g/mL Intermediate Hg Standard A: Into a 100 mL volumetric, pipet 500L Mercury Stock Standard (1000 ug/mL purchased standard). Bring to volume with deionized H20. Expires 1 week from preparation date Positive Control (100 ug/L or 150ug/L): To a 50 mL conical tube, add 2.0 mL of blank blood and 40L or 60L respectively of Hg Standard A and vortex to mix. External Control: such as BioRad Urine Metal Control Level 2: Place 2.0 mL of Urine Metal Control Level 2 into a 50mL conical tube.
VOLATLES BY GC
Critical Reagents ---Stocks, Standards, Controls, Calibrators: 0.012% n-Propanol (internal standard solution): Transfer 0.72 grams of n-propanol to a 6 liter volumetric lask and dilute to volume with distilled water. 17.3 mg/L Toluene standard: Transfer 20 ul toluene to a 1000 ml volumetric flask and dilute to volume with deionized water. Other standards: Calculate the concentration of the standard by taking a known volume of pure standard and diluting it to 1000 mL in deionized water. Multiply the density of the standard (g/ml or mg/ul) by the volume of standard (in microliters) to get the standard concentration in mg/L. The concentration may be calculated using the formula: Volume std * Density std / Final Volume std = Concentration std
13
VOLATILES BY MULTIPLE HEADSPACE

Critical Reagents ---Stocks, Standards, Controls, Calibrators: Standards should be prepared according to GC response of the compound of interest and the expected or suspected concentration of compound in the sample. Response will vary according to the compound of interest. Several standard preparations may be necessary before determining the appropriate concentration.
CARBOXYHEMOGLOBIN
10% Triton X-100 (v/v): In a 100 mL volumetric flask place 10 ml Triton X-100 into approximately 70 mL deionized water. Stir for ~ 1 hour. The solution may need to be slightly warmed or sonicated for complete dissolution. Bring to volume with deionized water. Expires One month after preparation date.
GUIDELINES FOR REAGENT CHECK BEFORE PLACING IN USE

Where applicable, a critical reagent should be run prior to use to ensure its reliability. Generally: 1. Run the new prepared reagent at the same time as the previous reagent and compare the results. 2. If there is any question about reagent reliability, seek supervisory assistance. 3. There can only be one of each type of critical reagent in use at a time. Be sure to record the Date In Use 4. Alk QCs need to be checked a minimum of two times by two different people.
14
Summary of Reagent Logbook Instructions 1. Critical reagents MUST be entered into the Reagent Logbook or the Response Factor Logbook 2. Prepared reagents that are listed in the Critical Reagents are assigned a Reagent Number 3. For reagents that are used as received from the manufacturer (ex Cerilliant Stocks, some BioRads), list the Date In Use and the lot number, but do not assign a Reagent Number. a. When a new or unique lot number is opened for use, it must be recorded. 4. Reagent numbers will be the date made (MMDDYY) and successive alphabetical letter. The alphabetical letter will restart at the start of each day. a. Ex. 011008A, 011008B, etc 011008AC,..(next day) 011108A, etc. 5. ALL items listed in the Reagent Logbook MUST have the Date In Use filled in. a. Enter either a date and an initial or Unsuitable and an initial. b. A Date In Use signifies that a solution has been tested and is suitable for analysis. c. If a solution is marked Unsuitable, then it has failed to meet analysis criteria. 6. Only ONE Critical Reagent of each type can be in use at any one time. a. When the Date In Use is filled out, any remaining older reagent should be disposed of. 7. The Reagent Number should be used to cross reference a solution in another location (ex. Response Factor Logbook) 8. Negative Controls consist of Blank Blood, DI Water, or Blank Urine. These items are either bought or tested prior to lab implementation. Discrepancies are always reported to a supervisor for evaluation before use. They are NOT recorded in the Reagent Log. Example Logbook Entries:
Reagent Number Date In Use/ Initials Name of QC/STD/ Reagent OPI,THC,COC ELISA LC and PC Description of Preparation Universal ELISA SOP 25uL OPI , 20uL THC, 10uL COC (LC) / 100uL OPI, 50uL THC, 50uL COC(PC) diluted to 1mL w/ blank blood Manufacturer and Lot Number 111107DF 111107DG 121207V Comments
011008A
unsuitable Initial________
Each Reagent Number used in this section Would be the OPI, THC and COC working stds.
REQUIRED to record when a new lot of a pure substance is opened.
(No Number)
01/10/08 AM Initial________ 0.08% EtOH Place the Cerilliant sticker here Or here
011008B
01/15/08 AM Initial________ 0.06mg/mL ALK QC
ALK SOP: 3.0mL each RF in 50mL, diluted w/MeOH
RF123107A RF123107B RF123107D RF123107G RF123107YY MeOH Baker 05487
Each Reagent Number used in this section Would be the methamphetamine, cocaine, lidocaine, citalopram, and trazodone 1.0 mg/ml response factors
15
CONTINUING IMPROVEMENT SECTION Any corrections, revisions, or ideas to the improvement of this document should be recorded here for consideration in the next official revision.
16
Response Factors
The numbering system for the Response Factors or Drug Stock Standards will consist of the letters RF followed by the date made (MMDDYY) and a successive alphabetical letter. The alphabetical letter will restart at the start of each day. Ex. RF011008A, RF011008B, RF011008AC,.. RF011108A, etc. Use the Reagent Number when cross referencing a solution in another location.
Response Factor Prep:

1.0 mg/mL Drug Stock Standard: Transfer 10 mg drug as the base to a 10 mL volumetric flask and dilute to volume with methanol. (Some drugs may require the addition of a drop of HCl to get complete dissolution.) If the drug is in the form of a salt or contains waters of hydration, the 10 mg must be adjusted accordingly. The calculation to convert the base to the salt is: mg of salt = mg of base x molecular weight of salt / molecular weight of base Note: Occasionally two molecules of drug are included in the salt form. Check the chemical formula prior to making standard. In this case, the molecular weight of the salt should be divided by 2 prior to using the formula above. Note: Make up the cocaine stock using acetonitrile instead of methanol. Since weighing 10.0 mg in a volumetric may be very difficult, weigh out an amount that is very close to 10.0 mg. Remember that 10.0 mg is the target and 11.0 mg or 9.0 mg would not be considered close. Once you have a weight, use that weight to calculate the actual concentration of the drug. amount weighed mg / 10.0 mL = standard concentration The 1.0 mg/mL concentration is the target value for most drugs but may vary for some.
Evaluation:
Generally: 1. Run the new prepared response factor and compare the results to previous. 2. If there is any question regarding response factor reliability, seek supervisory assistance. 3. Review the C/A ratio, anything outside 0.44 and 0.75 should be checked by supervisor 4. The new response factors should be within +/- 10% of the old response factor; if not, advise a supervisor.
Response Factor Instructions Version 2.0
CONTINUING IMPROVEMENT SECTION Any corrections, revisions, or ideas to the improvement of this document should be recorded here for consideration in the next official revision.
Response Factor Instructions Version 2.0

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