Toxicology Manager Document Type: LC & LC/MS/M
Harris County Institute of Forensic Sciences Approved by: Toxicology Section: Toxicology Manager Document Type: LC & LC/MS/MS Procedure No.: TOX.LC.3496
Title: Bath Salts Confirmation by LCMSMS Rev.: 7
1.0 Purpose 1.1 This assay is used to qualitatively identify certain synthetic cathinones, amphetamine-like compounds, known as “Bath Salts” by LC-MS/MS.
2.0 Scope
2.1 The method is intended for finding evidence of “Bath Salt” compounds in blood and urine specimens, as well as other fluids and tissues. The method specifically scans for mephedrone, methedrone, methylone, butylone, ethylone, methylene- dioxypyrovalerone (MDPV), naphyrone, alpha-pyrrolidinopropiophenone (α- PPP), alpha-pyrrolidinopentiophenone (α-PVP), pentedrone, and 4'-Methyl-α- pyrrolidinopropiophenone (Methylpyrrolidinopropiophenone,4-MePPP, M-PPP). Other bath salt compounds and 2-propanal variants are possible, but not detected by this method.
3.0 Definitions and Abbreviations
3.1 No method-specific or non-standard terms are used in this procedure.
4.0 Materials
4.1 Instruments and Equipment
4.1.1 Pipettes and tips 4.1.2 13 x 100 mm screw cap test tubes and caps 4.1.3 16 x 100 mm test tubes 4.1.4 Vortex mixer 4.1.5 Centrifuge 4.1.6 Nitrogen stream evaporator and compressed nitrogen gas 4.1.7 Autosampler vials and septum caps 4.1.8 Filter and disposable syringes 4.1.9 Agilent LCMSMS
4.2 Reagents
4.2.1 Acetonitrile: HPLC Grade.
4.2.2 Water: HPLC Grade.
4.2.3 Ethyl Acetate: HPLC Grade
Page 1 of 8 Harris County Institute of Forensic Sciences Approved by: Toxicology Section: Toxicology Manager Document Type: LC & LC/MS/MS Procedure No.: TOX.LC.3496
Title: Bath Salts Confirmation by LCMSMS Rev.: 7
4.2.4 Methanol: HPLC Grade
4.2.5 pH 12 Saturated Sodium Borate Buffer A. Weigh 100 g of Sodium Tetraborate B. Add to 900 mL of deionized water. C. Heat with stirring bar in a 1 L beaker until dissolved. D. Add 50 mL of 10 N Sodium Hydroxide while stirring, allow to cool. E. Adjust to pH 12 + 0.2 with 10 N NaOH. F. Stable 6 months at room temperature.
4.2.6 Mobile phase A A. Measure 1 L of deionized water into a clean 1000 ml Media bottle. B. Add 0.45g of ammonium formate to the 1 L of Millipore Milli Q or HPLC grade water. C. Mix well. D. Stable for 3 months at room temperature. E. Label as 7 mM Ammonium formate in H2O (Mobile Phase A). Include preparation and expiration date (3months) and initials of preparer. F. Stable at room temperature.
4.2.7 Mobile phase B: Acetonitrile. A. Stable for 3 months at room temperature.
4.2.8 Reconstitution buffer (90:10 7mM Ammonium Formate in H2O:ACN) A. Add 5 mL of acetonitrile to 50 mL conical vial. B. Add 45 mL of 7mM ammonium formate (mobile phase buffer A used in benzodiazepine methods). C. Cap and mix by inversion or vortexing. D. Stable for 3 months at room temperature.
4.3 Stock Standards
Note: Certified Reference Materials should be used within manufacturer’s expiration date and be stored at the recommended storage temperature.
4.3.1 Butylone-D3 HCl, 100 µg/mL from Cerilliant #B-046 or equivalent 4.3.2 Butylone HCl, 1.0 mg/mL from Cerilliant #B-045 or equivalent 4.3.3 Ethylone HCl, 1.0 mg/mL from Cerilliant #E-071 or equivalent 4.3.4 Mephedrone HCl, 1.0 mg/mL from Cerilliant #M-138 or equivalent 4.3.5 Methylone HCl, 1.0 mg/mL from Cerilliant #M-140 or equivalent 4.3.6 MDPV HCl, 1.0 mg/mL from Cerilliant #M-146 or equivalent
Page 2 of 8 Harris County Institute of Forensic Sciences Approved by: Toxicology Section: Toxicology Manager Document Type: LC & LC/MS/MS Procedure No.: TOX.LC.3496
Title: Bath Salts Confirmation by LCMSMS Rev.: 7
4.3.7 Methedrone HCl, 1.0 mg/mL from Cerilliant #M-147 or equivalent 4.3.8 Naphyrone HCl, 1.0 mg/mL from Cerilliant #N-067 or equivalent 4.3.9 Pentedrone HCl, 1.0 mg/mL from Cerilliant #P-087 or equivalent 4.3.10 α-PPP 1.0 mg/mL from Cayman # 10445 or equivalent 4.3.11 α-PVP 1.0 mg/mL from Cayman # 9001083 or equivalent 4.3.12 M-PPP 1.0 mg/mL from Cayman # 10446 or equivalent
4.4 Working Standards / Controls
Note: Prepared working standards/QCs may not have an expiration date that exceeds the expiration date of the stock standard used to prepare it.
Note: Prepared working internal standards/standards/controls are stable until the manufacturer’s expiration date when stored at manufacturer’s recommended storage temperature.
4.4.1 Bath Salts Control (A) 5 µg/mL A. Pipette 25 µL of the following stocks into a 5 mL volumetric flask: a. Mephedrone b. Methedrone c. Methylone d. Butylone e. Ethylone f. MDPV g. Naphyrone B. QS to 5 mL with Methanol. C. Label as Bath Salts Control (A). Include name of preparer, date prepared, expiration date, and concentration.
4.4.2 Bath Salts Control (B) 5 µg/mL A. Pipette 25 µL of the following stocks into a 5 mL volumetric flask: a. Pentedrone b. α-PPP c. α-PVP d. M-PPP B. QS to 5 mL with Methanol. C. Label as Bath Salts Control (B). Include name of preparer, date prepared, expiration date, and concentration.
4.4.3 Bath Salts Control (C) 0.5 µg/mL A. Pipette 2.5 mL of Control A and 2.5 mL of Control B into a 25 mL Page 3 of 8 Harris County Institute of Forensic Sciences Approved by: Toxicology Section: Toxicology Manager Document Type: LC & LC/MS/MS Procedure No.: TOX.LC.3496
Title: Bath Salts Confirmation by LCMSMS Rev.: 7
volumetric flask B. QS to 25 mL with Methanol C. Label as Bath Salts Control (C). Include name of preparer, date prepared, expiration date, and concentration.
4.4.4 Bath Salts Internal Standard 1 µg/mL A. Pipette 100 µL of the stock 100 µg/mL Butylone-D3 into a 10 mL volumetric flask. B. QS to 10 mL with methanol. C. Using a white color label, mark as Bath Salts Internal Standard. Include name of preparer, date prepared, expiration date, and concentration.
5.0 Procedure
5.1 Standards and Controls
5.1.1 Label a sufficient number of 13 x 100 mm screw cap tubes.
5.1.2 Add the indicated volumes (µL) of Working Controls.
Tube contents Target QC C I.S. (ng/mL) (0.5 µg/mL) (1 µg/mL) Reagent Blank ------Negative QC ------5 QC 1 100 100 5 QC 2 5 5 5
5.1.3 Add 5 μL of the Working Internal Standard solution to all tubes except the reagent blank.
5.2 Sample Preparation and Extraction
5.2.1 Using labeled 13 X 100 mm screw cap tubes, pipet 0.5 mL of negative blood into standard/QC tubes and 0.5 mL specimen into case tubes.
5.2.2 Add 0.2 mL pH 12 saturated sodium borate and vortex for 5 seconds.
5.2.3 Add 2 mL of ethyl acetate and vortex for 5 seconds.
Page 4 of 8 Harris County Institute of Forensic Sciences Approved by: Toxicology Section: Toxicology Manager Document Type: LC & LC/MS/MS Procedure No.: TOX.LC.3496
Title: Bath Salts Confirmation by LCMSMS Rev.: 7
5.2.4 Centrifuge at 3500 RPM for 5 minutes.
5.2.5 Transfer organic layer into a fresh 16 x 100 mm tube.
5.2.6 Evaporate solvent under nitrogen gas stream.
5.2.7 Reconstitute in 1.0 mL reconstitution buffer (90% of 7mM Ammonium Formate in H2O and 10% of Acetonitrile).
5.2.8 Filter specimen with filters attached to disposable syringes
5.2.9 Transfer filtrate to autosampler vials
5.2.10 Analyze samples by LCMSMS
5.3 Instrument Parameters
5.3.1 Table 2: Mobile Phase A 7mM Ammonium Formate in Water B Acetonitrile
5.3.2 Table 3: LC Conditions Internal Particle Column Length Column Flow Diameter Size C18 100 mm 3 mm 3.5µm 0.25 mL/min
HPLC Program (Gradient) Time (min.) %A %B 0 80 20 3 80 20 4 5 95 7.49 5 95 7.5 80 20 14 80 20
5.3.3 Table 4: QQQ Conditions Collision Precursor Product Ions Compound Energy Ion (Quant/Qual) (V) Naphyrone 282.2 126 25
Page 5 of 8 Harris County Institute of Forensic Sciences Approved by: Toxicology Section: Toxicology Manager Document Type: LC & LC/MS/MS Procedure No.: TOX.LC.3496
Title: Bath Salts Confirmation by LCMSMS Rev.: 7
155 25 126 25 MDPV 276.1 135 25 174 15 Ethylone 222.2 204 10 174 15 Butylone 222.1 204 10 160 15 Methylone 208.1 132 30 176 5 Methedrone 194.1 161 20 160 10 Mephedrone 178.1 144 35 177 15 Butylone-D3 225.1 207 10 174 8 Pentedrone 192.2 91.1 20 105.1 24 α-PPP 204.1 98.1 24 91.1 24 α-PVP 232.2 77.1 55 119.1 24 M-PPP 218.2 147.1 16
5.3.4 Start sequence with a blank mobile phase injection using the pre-run method to equilibrate the column for bath salts method.
A. Table 5: LC Condition (Bath Salts Pre-run.m) HPLC Program (Gradient) Time (min.) Flow (mL/min) %A %B 0 0.3 80 20 2 0.3 5 95 5 0.3 5 95 6 0.3 80 20 16 0.3 80 20 17 0.3 5 95 20 0.3 5 95 21 0.3 80 20 30 0.3 80 20
Page 6 of 8 Harris County Institute of Forensic Sciences Approved by: Toxicology Section: Toxicology Manager Document Type: LC & LC/MS/MS Procedure No.: TOX.LC.3496
Title: Bath Salts Confirmation by LCMSMS Rev.: 7
5.4 Table 5: Sample Injection Sequence
Position Sample Number 1 MPB – Pre-Run Method 2 Reagent Blank 3 Negative control 4 Control 1 3 Negative control (reinjection) 5 Continue with samples Run the extracted (5ng/mL) positive control at the end of the run. If the run contains more than 20 specimens, another re- injection of the positive control must be in the middle of the run
6.0 Data Analysis/Interpretation/Documentation
6.1 Refer to SOP: TOX.LC.3488 Agilent 6460 Triple Quad Processing Quantitative Data
6.2 Use the 100 ng/mL QC as a single calibrator forced through zero
6.3 Use processed data to fill out analyte verification form TOXF.073
7.0 Acceptance Criteria
7.1 Refer to SOP: TOX07.3408 LC/MS/MS Analyses, Quality Control, Acceptance and Reporting Criteria
7.2 All spiked drugs in the QCs need to be present with good quality peak and ion ratio. The semi-quantitation result of all the drugs in the 5 ng/mL QC should read greater than 1 ng/mL for the run to be acceptable.
7.3 The administrative cut-off is 1 ng/mL. Cases results less than the 1 ng/mL are reported as “None detected.”
7.4 Cases with confirmation results that are above 1 ng/mL should be reported as “Present.”
8.0 References
8.1 Péter Jankovics, András Váradi, László Tölgyesi, Szilvia Lohner, Júlia Németh- Palotás, Hilda Kőszegi-Szalai, “Identification and characterization of the new Page 7 of 8 Harris County Institute of Forensic Sciences Approved by: Toxicology Section: Toxicology Manager Document Type: LC & LC/MS/MS Procedure No.: TOX.LC.3496
Title: Bath Salts Confirmation by LCMSMS Rev.: 7
designer drug 4′-methylethcathinone (4-MEC) and elaboration of a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) screening method for seven different methcathinone analogs” Forensic science international 15 July 2011 (volume 210 issue 1 Pages 213-220 DOI: 10.1016/j.forsciint.2011.03.019)
8.2 SOP: TOX09.3470 Agilent 6460 Triple Quad System Startup and Shutdown
8.3 SOP: TOX.LC.3484 Agilent 6460 LC/Triple Quad Daily Maintenance, Tuning, and Calibration
8.4 SOP: TOX09.3469 Agilent 6460 Triple Quad Data Acquisition
8.5 SOP: TOX07.3409 LC/MS/MS Chromatographic Peak Check
9.0 Revision History
Revision Description of Change Reviewed By Date 0 Original SOP J. Walterscheid 10/11 Updated concentration of working internal standard, 1 clarified the pH of the saturated borate buffer A. Beard 0112 Added drugs of Pentedrone. A-PPP, a-PVP and M-PPP 2 and changed title to confirmation M.Chen 0712 Add butylone-D3 to standard list; update standard/control expiration date criteria, added 3 administrative cut-off CA 1212 Added the administrative cutoff, “or equivalent” wording and re-injection of positive controls; removed non-critical instrument parameters; uniform working F. Guale/ 4 control name; A. Beard/CA 1013 Add working QC C to make new 5 ng/mL QC; Add 5 5 ng/mL QC acceptance criteria CA 1213 6 Correct QQQ Table; reference TOXF.073 CA 1114 0415; 7 Add pre-run method; “in water” Table 2 CA 0615
10.0 Attachment(s) or Appendices
10.1 Not applicable
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