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Technical Report 2017 Collaborative Study on Ammonia and Benzo[A]

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Technical Report 2017 Collaborative Study on Ammonia and Benzo[A] Tobacco and Tobacco Products Analytes Sub-Group Technical Report 2017 Collaborative Study on Ammonia and Benzo[a]pyrene in Tobacco Products March 2018 Study Project Leaders: Regina Ballentine, Altria Client Services LLC, U.S.A. Anthony Brown, Altria Client Services LLC, U.S.A. Author: Regina Ballentine, Altria Client Services LLC, U.S.A. Co-Author and Statistical Analysis: Michael Morton, Ph.D., Altria Client Services LLC, U.S.A. Table of Contents 1. SUMMARY ...................................................................................................................... 3 2. INTRODUCTION ............................................................................................................ 3 2.1 Objective .................................................................................................................. 3 3. ORGANISATION ............................................................................................................ 3 3.1 Participants .............................................................................................................. 3 3.2 Protocol .................................................................................................................... 4 4. DATA – RAW .................................................................................................................. 6 5. DATA – STATISTICAL ANALYSIS ............................................................................. 6 5.1 Exclusion of Outliers ............................................................................................... 6 5.2 Calculation of Repeatability (r) and Reproducibility (R) ........................................ 7 6. DATA INTERPRETATIONS .......................................................................................... 8 7. RECOMMENDATIONS .................................................................................................. 9 8. APPENDICES .................................................................................................................. 9 1. Summary At the May 2017 CORESTA Tobacco and Tobacco Products Analytes Sub-Group (TTPA) meeting held in Charlottesville, USA, the Sub-Group initiated a collaborative study for the determination of ammonia and benzo[a]pyrene in smokeless tobacco, cigarette filler, and ground cigars (wrapper, binder, and filler). The intent of this study was to support laboratory accreditation and to expand the scopes of CORESTA Recommended Methods (CRMs) N° 79 and N° 82 to include ground cigars. 2. Introduction At the CORESTA Tobacco and Tobacco Products Analytes Subgroup (TTPA) meeting held in Charlottesville, USA (May 2017), it was decided to conduct a collaborative study for Ammonia and Benzo[a]pyrene (B[a]P) using the following CRMs: CRM N° 79, Determination of Ammonia in Tobacco and Tobacco Products by Ion Chromatographic Analysis (April 2015) CRM N° 82, Determination of Benzo[a]pyrene in Tobacco Products by GC-MS (July 2017) The collaborative study involved 17 laboratories and will support laboratory accreditation and a scope expansion of the CRMs to include ground cigars (wrapper, binder, and filler). Tabulated data are presented along with repeatability (r) and reproducibility (R). The results of the study demonstrate that the CRMs for Ammonia and B[a]P are suitable for the analysis of ground cigars. The TTPA recommends that the two CRMs be updated to include ground cigars. 2.1 Objective This study was conducted to support laboratory accreditation and scope expansion of the Ammonia and B[a]P CRMs beyond just tobacco, cigarette filler, and smokeless tobacco to include ground cigars. This work was conducted using the samples listed in Table 2. Data were collected from the participating laboratories and statistically evaluated in basic conformance with the recommendations of ISO 5725-2:1994 and ISO/TR 22971:2005. 3. Organisation 3.1 Participants A list of the participating laboratories is provided in Table 1. Not all laboratories provided data for both methods nor did all laboratories analyze all samples. The laboratories are listed in alphabetical order. Identifying number codes were assigned to each laboratory to protect confidentiality and do not correspond to the order shown in the table below. TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 3/29 Table 1: List of Participating Laboratories in the 2017 Collaborative Study Participating Laboratories Ammonia B[a]P Altria Client Services LLC, United States X X American Snuff Company, United States X C.I.T. Montepaz S.A., Uruguay X X China National Tobacco Quality Supervision and Test Center, X X China CNTC, Zhengzhou Tobacco Research Institute, X X Key Laboratory of Tobacco Flavor Basic Research, China CNTC, Yunnan Tobacco Quality Supervision and Test Station, X X China Enthalpy Analytical, Inc., (Richmond) United States X X Global Laboratory Services, United States X X ITG Brands, United States X X Japan Tobacco Inc., Japan X X JTI-Ökolab, Austria X KT&G Research Institute, South Korea X X Imperial Tobacco, Reemtsma, Germany X RJ Reynolds Tobacco Company, United States X X Swedish Match, North America, United States X Swedish Match, Northern Europe, Sweden X University of Kentucky, United States X X 3.2 Protocol Specific details from the protocol are described below: 3.2.1 Sample Shipment Laboratories were responsible for procuring 1R6F ground cigarette filler from the University of Kentucky, CRP1.1, CRP2.1, CRP3.1, and CRP4.1 from North Carolina State University, and Cigar filler #1-05/17 and Cigar M16-05/17 from Altria Client Services LLC. Laboratories were requested to store the samples at approximately 4 °C upon receipt. If samples were not analysed within one week, they were to be stored in a freezer at approximately −20 °C until the analyses were performed. Laboratories were requested to conduct the study July through August and report data by August 15, 2017. The samples are identified in Table 2. The protocol incorrectly identified Cigar Filler #1-05/17 as only ground filler; however, this sample was a ground finished cigar that included the wrapper, binder, and filler. TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 4/29 Table 2: Sample Identification Product Type CRP1.1 - Swedish-style snus pouch CRP2.1 - American-style loose moist snuff CRP3.1 - American-style loose dry snuff powder CRP4.1 - American-style chopped loose-leaf chewing tobacco 1R6F Ground Filler-RT1 - American blend cigarette filler Cigar Filler #1-05/17 - Flavoured ground cigar (wrapper, binder, and filler) Cigar M16-05/17 - Traditional dark-air cured ground cigar (wrapper, binder, and filler) 3.2.2 Within Laboratory Sample Preparation The laboratories were directed to remove samples from the −20 °C freezer and place the unopened samples in a refrigerator for a minimum of 24 hours to ensure that the water in the sample had been fully equilibrated. Samples could then be removed from the refrigerator for a minimum of 2 hours prior to opening for analysis. Once samples were opened, the samples could be stored in a tightly sealed container at approximately 4 ºC for up to one week. Additional handling requirements are described below: CRP1.1: The Snus pouches should be cut into 2 halves and both parts added directly into the extraction vessel. Both Snus and pouch material (paper) are to be analyzed. All other samples should be mixed with a spatula before removing aliquots for analysis. 3.2.3 Sample Analysis and Data Reporting The participating laboratories were instructed to conduct three replicate analyses (individual tobacco weighing) for Ammonia and B[a]P using the current versions of the CRMs. Ammonia: CRM N° 79, Determination of Ammonia in Tobacco and Tobacco Products by Ion Chromatographic Analysis (April 2015) B[a]P: CRM N° 82, Determination of Benzo[a]pyrene in Tobacco Products by GC-MS (July 2017) Participating laboratories were requested to document any deviations from the protocol and the CRMs and submit the deviations with their results. As stated in the protocol, data submitted with significant deviations from the applicable CRM would be excluded from the study. Deviations reported by the laboratories are identified below. The following deviations for Ammonia CRM N° 79 were reported: Lab 1 – 1) No suppressor was used. 2) Elution was done with 3 mM Oxalic acid solution containing 1 mM 18-Crown-6 ether. Lab 2 – A segmented flow analyzer was used for analysis. Lab 3 – The amount of sample and volume of extraction solution was changed. Lab 5 – A chilled auto-sampler tray was not used. Lab 8 – No suppressor was used. Lab 14 – 1) Modified the mobile phase gradient because a CS19 column was used. 2) Several sample dilutions were performed prior to analysis. TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 5/29 The following deviations for B[a]P: CRM N° 82 were reported: Lab 1 – 1) ultrasonic extraction was used instead of an orbital shaker or wrist action shaker. 2) Hexane was used for calibration standards instead of 50:50 toluene:isooctane. 3) There was no filtration of the sample extract 4) Evaporated solvent before washing the SPE cartridge 5) Hexane was the only wash solvent used for solvent phase extraction even though methanol: water and isopropyl alcohol were also specified. Lab 2 – Used a DB-5MS (30 m x 0.25 mm x 1.0 µm) column. Lab 10 – Used a DB-35MS column. Lab 11 – Used a DB-35MS column. Lab 12 – 1) Used a DB-5MS Ultra Inert column. 2) Concentration differed from
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