TOXICOLOGY LABORATORY OFFICE OF CHIEF MEDICAL EXAMINER CITY OF NEW YORK SCREENING FOR BASIC DRUGS BY GAS CHROMATOGRAPHY WITH SIMULTANEOUS NITROGEN-PHOSPHORUS AND MASS SPECTROMETRY DETECTION PRINCIPLE Basic drugs encompass the largest group of compounds analyzed at the OCME FTL. This procedure is designed to screen basic drugs in biological specimens using simultaneous analysis by gas chromatography (NPD) and full scan mass spectrometry (GCMS). The procedure is used for qualitative analysis. Solid phase extraction (SPE) is used to extract basic drugs from biological fluids or tissue homogenates. In SPE, the drug is temporarily bound to a sorbent in the solid phase column as the prepared sample is poured through these columns. The column is washed to remove interfering compounds, followed by elution of the drug. The eluent is evaporated and then reconstituted. The resulting solution is analyzed by NPD/GCMS. A single point calibration curve is used for screening analysis along with a negative control (blank) and a 0.2 mg/L positive control. The multi-component calibrator and control contain analytes frequently encountered in this laboratory. SAFETY The handling of all samples, reagents and equipment is performed within the established laboratory safety guidelines detailed in the safety manual. REAGENTS AND MATERIALS All chemicals should be analytical reagent (AR) grade or higher. The chemical reagents required for the extraction procedure are prepared as indicated below. Each prepared reagent is stable for a minimum of six months. All new reagents are validated against the old ones prior to use. 1. Deionized water (DI H 2O) 2. Negative Matrices a. Certified negative matrices (blood, brain, and liver from calf or sheep) are obtained from outside source and validated in-house. These are stored frozen (-10 oC or lower). 3. Sodium acetate trihydrate . Certified ACS. Fisher Scientific or equivalent. 4. 100mM sodium acetate buffer (pH 4.5) a. Dissolve 11.72g sodium acetate trihydrate in 400mL DI H 2O; add 6.48mL glacial acetic acid. Dilute to 2L using DI H 2O. Mix. Adjust pH to 4.5 ± 0.1 with sodium acetate or acetic acid. Note that this volume may be scaled up or down depending upon the needs of the laboratory. Page 1 of 28 b. Storage: in glass; room temperature. c. Stability: 6 months; inspect daily before use for contamination. 5. pH 9.0 buffer a. Dissolve 40g KHCO 3 in 800mL DI H 2O; add 20g K 2CO 3. Dilute to 2L using DI H2O. Mix. Adjust pH to 9.0 ± 0.1 with KHCO 3 to lower the pH, or K 2CO 3 to raise the pH . b. Storage: room temperature in glass. c. Stability: 6 months. Inspect for contamination each day of use. 6. HCl, concentrated, 12.1N . Fisher Scientific or equivalent. 7. Hydrochloric Acid, 100mM a. Add 16.8mL concentrated HCl to 800mL Deionized water. Dilute to 2000mL with deionized H 2O. b. Store at room temperature in glass or plastic. c. Stability: 6 months. Inspect each day before use for contamination. 8. Methanol (Fisher Scientific - ACS Certified) 9. Ethyl Acetate , Fisher Scientific Optima or equivalent. 10. Ammonium Hydroxide, NH 4OH (Fisher Scientific) 11. Eluting solvent: Ethyl acetate/NH 4OH (98/2). Prepare fresh daily for use. Note: Ammonium hydroxide will break down to ammonia and water. The ammonia will evaporate if the container is not kept closed. This will cause a pH decrease, making the reagent unsuitable for solid phase extraction. Use small lots of working solution (3 mL of ammonium hydroxide in a 6 mL crimp top vial. Use once then discard the remaining), open a fresh bottle daily. Always keep ammonium hydroxide refrigerated. 12. Polycrom Clin II Solid Phase Extraction Columns , CEREX 13. System 48 Processor connected to nitrogen source. 14. Waste Rack, SPE Rack, Collection Tube Rack. 15. Concentrator (Turbovap or SPEware) connected to a nitrogen source. 16. Vacuum Filtration Apparatus 17. Toluene/Heptane/Isoamyl Alcohol, 39:10:1(THI) a. Add 780mL of toluene, 200mL of heptane and 20mL of isoamyl alcohol to a 1000mL graduated cylinder. Stir for approximately one hour, and then transfer to a 1L repipeter. Page 2 of 28 SPECIMEN PREPARATION On occasion the FTL will receive specimens that may be of poor quality; these will be flagged as unsuitable for testing in both the appropriate case file and in DataEase. For example, decomposed specimens may exhibit the following characteristics: presence of green, brown, and black colors; layer(s) of fat; oily mixture in the sample and foul sample smell. Decomposition can affect results of testing so it is important to choose the most suitable specimen for testing. Characteristics of decomposed samples are not limited to what is listed above, the Medical Examiner may have noted that the specimen is decomposed and to what extent of this decomposition on the Toxicology Request form. If there is any question as to whether a sample is decomposed consult with a supervisor. The procedure is routinely applied to the following biological specimens and their aliquots unless otherwise specified: Ensure samples are homogenous; if necessary homogenize the specimen prior to aliquoting Blood 1 mL of the undiluted specimen Urine 1 mL of an undiluted specimen Bile 1 mL of a 1:5 dilution (1 mL of undiluted bile qs to 5 mL with deionized water) Brain 1 mL of a 1:3 homogenate (5 g homogenized with 10 mL of deionized water) Liver 1 mL of a 1:5 homogenate (5 g homogenized with 20 mL of deionized water) Gastric 1 mL of a 1:10 homogenate (For solid samples, homogenize 2 g with 18 mL of deionized water otherwise dilute 2 mL of thoroughly mixed gastric and q.s. to 20 mL.) Note : Do not use homogenates older than two weeks unless low sample size requires it. Discuss with supervisor and note in case record. Note: The entire submitted amount of gastric contents must be weighed prior to homogenizing and sampling; total weight must be documented on reports. EXTRACTION PROCEDURE 1. Print a worklist of cases to be analyzed. Review with supervisor for any updates of priority (RUSH) cases or other special instructions. Batches should contain 20-25 cases plus the associated calibrator and controls. This batch size limit may be increased by Page 3 of 28 authority of the section supervisor as needed. 10% of the batch samples are to be QC specimens. 2. Retrieve all samples on the worklist and place in an empty rack. Allow samples to come to room temperature. Note : If for any reason there is a discrepancy with the sample listed on the worklist for extraction check the appropriate case folder for any possible notations, if there is no information make a note in Data Ease, on the appropriate case analysis form, and ensure a discrepancy form was submitted for the sample. Consult with a supervisor as necessary. i.e. sample listed on the worklist for extraction is subclavian blood but the sample submitted is not labeled or is labeled as another source i.e. sample has low volume (QNS); may want to schedule a different sample for testing 3. Obtain enough 16 x 125 mm culture test tubes to extract one negative control, calibrator, positive controls and all requested case work. Label the tubes appropriately. Tubes should bear the entire toxicology number (e.g., 06-1234, not 1234), the specimen type and any appropriate dilution. 4. Pipet 1.0 mL of an appropriate negative matrix into each of the controls and calibrator tubes. Refer to the REAGENTS AND MATERIALS section of this document for specific matrix for the analysis. 5. Next, mix each specimen thoroughly by gently inverting container two to four times. Pipet 1.0 mL of each specimen into the appropriately labeled tube. Make certain that the toxicology number on the specimen bottle exactly matches the number on the tube. If a blood specimen is being pipetted and a clot is encountered, homogenize the specimen using either the Polytron Homogenizer or a glass hand homogenizer; document on the blood bottle that the specimen was homogenized, date and initial the label and annotate on sequence list. Continue in this fashion until all cases have been pipetted. Note: Immediately refrigerate all case specimens and negative matrices. Do not leave this for a later time. 6. Remove all extracted cases in the DataEase database so the samples are not duplicated by another analyst. CAUTION : DO NOT WAIT UNTIL THE FOLLOWING DAY TO COMPLETE THE ABOVE! 7. Prepare calibrator by fortifying the appropriate tube with various solutions as indicated: a. 1.0 mL blood aliquots: Add 10 µL of 100 mg/L Basic Drugs Group 1 Calibrator to the Cal 1 - 1.0mg/L tube only. b. 1.0 mL urine aliquots: Add 10 µL of 100 mg/L Basic Drugs Group 1 Calibrator to the Cal 1 – 1.0mg/L tube only. Page 4 of 28 c. 1.0 mL tissue aliquots: Add 10 µL of 100 mg/L Basic Drugs Group 1 Calibrator to the Cal 1 – 1.0mg/L tube only. 8. For urine screen batches prepare the 0.2 mg/L control (QC) by fortifying the QC 1 – 0.2 mg/L tube with 20 µL of 10 mg/L Basic Drugs Group 1 Control in 1.0 mL of deionized water. 9. For blood screen batches, obtain an appropriately labeled external 0.2 mg/L control (QAS) from the freezer and allow it to come to room temperature. Aliquot 1.0 mL of the QAS control into the appropriately labeled test tube. 10. For tissue screening, prepare the 0.2 mg/L control (QC) by fortifying the QC 1 – 0.2 mg/L tube with 20 µL of 10 mg/L Basic Drugs Group 1 Control in 1.0 mL of an appropriate negative matrix.