SEP 1 8 2001
Final
Sample Analysis Protocol
Study Title
Determination of Caffeine and Caffeine
Metabolites in Human Urine Samples from PM
Project No. 1148, Covance CRU Study No.
12226-8450 using Liquid-Liquid Extraction with
HPLC Detection
Study Director
Mike Klawikowski
Sponsor
Philip Morris U.S.A.
4201 Commerce Road
GateC, Door 17
Richmond
Virginia 23234
Sponsor's Contact
Robin Kinser, PhD
Test Facility
Covance Laboratories Inc.
3301 Kinsman Boulevard
Madison
Wisconsin 53704
USA
Proposed Dates
Analysis Start Date:
Analysis End Date:
12 September 2001
17 September 2001
Covance Study Number
7209-114
Sponsor Reference
1148
Page Number
1 of 5
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Covance Study Number 7209-114
Final Sample Analysis Protocol
GENERAL COVANCE REQUIREMENTS
Purpose of Study
The objective of the study is to determine concentrations of caffeine (137X) and its
metabolites, 1,7-dimethylxanthine (17X), 1,7-dimethyluric acid (17U), 1-methylxanthine
(IX), and 5-acetylamino-6-formylamino-3-methyluracil (AFMU) in human urine using
liquid-liquid extraction with HPLC detection, for Philip Morris, USA, Study Reference
Number 1148, entitled "A Single-Center Pilot Exploratory Study to Evaluate Study
Design, Study Logistics, and Bioanalytical Methods to Measure Smoke Exposure in Adult
Smokers of 3.0 to 6.9 mg FTC Tar Delivery Cigarettes as Compared to Adult NonSmokers (Pilot Total Exposure Study)." This analysis will utilize the analytical method
described in MP-CMET-MA, previously validated at Covance Laboratories Inc. under
Covance Study Number 7209-109.
Reference Materials and Matrix
137X, Sigma Chemical Company
17X, Sigma Chemical Company
17U, Sigma Chemical Company
IX, Sigma Chemical Company
AFMU, Toronto Research Chemicals
Vanillic Acid, internal standard, Sigma Chemical Company
Reference materials will be handled according to Covance standard operating procedure
(SOP) NA-BAC. 205. The supplier, lot numbers, storage conditions, and expiration date
will be retained at Covance.
Human urine, adjusted to pH 3.5, approximately, by the addition of 20 mg/mL ascorbic
acid at time of collection, will be obtained from in-house collections at Covance. The
source, lot number, and storage conditions will be documented in the raw data.
Samples
A total of 140 samples is scheduled for analysis, to be provided by Covance Central
Laboratory Services (CCLS). Samples will be handled according to Covance SOPs NABAC. 100 and NA-BAC. 102, as applicable. Samples will be stored at -20°C until
analysis. Remaining samples will be maintained at Covance in a freezer set to maintain
-20°C for 90 days after issuance of the draft report, pending further notification by the
Sponsor of further sample disposition.
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Assay Criteria
Acceptance of each analytical run is based on Covance SOP NA-BAC. 211.
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Back-calculated concentrations for calibration standards must be <±15.0% from
theoretical (<±20.0% at the LLOQ). No more than 25% of the calibration standards
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Covance Study Number 7209-114
Final Sample Analysis Protocol
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may be excluded and at least six non-zero calibration standards must remain in the
calibration curve for it to be acceptable.
Each analytical run will be acceptable if at least half of the quality control (QC)
samples at each concentration and two-thirds of all QC samples in the curve range are
within 15% of theoretical.
Dilution QC samples will be included, at least in duplicate, for each analytical run
containing samples requiring dilution. These QC samples will be diluted with blank
matrix to the same extent as the most highly diluted sample, and will be included at
least at every order of magnitude utilized for dilutions. At least half of the dilution QC
samples must be within 20% of theoretical for the results of diluted samples to be
accepted.
Study samples will be routinely reassayed only when values exceed the curve range
or when the analyst has identified analytical problems.
Any additional reassays requested by the Sponsor or Principal Investigator will be
run in duplicate (where sufficient volume allows), with the reported value determined
as detailed in Covance SOP NA-BAC. 211. The reason for these reassays will be
documented in the raw data.
Analysis Report
Upon completion of assays, a draft report will be supplied to the Covance Quality
Assurance Unit (QAU) and the Sponsor for review and comment. The report will be
finalized after incorporation of QA and Sponsor comments. Descriptive statistics (means,
standard deviations, relative standard deviations, and deviations from theoretical) will be
calculated for the calibration standards and QC samples.
Archive Statement
All raw data, documentation, records, sample analysis outline, and an original signed copy
of the final report will be archived in the storage facilities of Covance. These materials
will be retained by the Covance site at which the work is performed. At least 1 year after
submission of the final report, the Covance archive staff will contact the Sponsor, and the
aforementioned materials will be sent to the Sponsor, and a return fee will be charged;
with the exception that the raw data stored on magnetic media, sample analysis outline,
study correspondence, and an original signed copy of the final report will be retained by
Covance.
REGUIATORY REQUIREMENTS
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This analytical study is intended for the support of a human clinical study. The procedures
will be performed in compliance with:
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OECD Principles on Good Laboratory Practice (revised 1997, Issued Jan 1998)
ENV/MC/CHEM(98)17
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Covance Study Number 7209-114
Final Sample Analysis Protocol
United States Food and Drug Administration Title 21 Code of Federal Regulations
Part 58. Good Laboratory Practice for Nonclinical Laboratory Studies Issued 22
December 1978, Federal Register, plus subsequent amendments.
The study described in this protocol will be subject to Quality Assurance evaluation. The
form of inspection is described in the Quality Assurance Unit (QAU) SOPs. Unless
otherwise stated, the in-lab performance, data andfinalreport generated as a result of this
study will be audited by QAU of Covance in accordance with applicable Covance SOPs.
Any deviations or changes to the above will be documented in the raw data and
summarized in thefinalreport. The impact of all such occurrences on sample analysis will
be evaluated by the Study Director.
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Covance Study Number 7209-114
Final Sample Analysis Protocol
APPROVAL
The Sponsor's Contact approved of this sample analysis outline on August 30, 2001 via
electronic mail.
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Mike Klawikowski
Study Director
Covance Laboratories Inc.
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Daniel F. Kehoe
Manager, Pharmaceutical Analysis
Covance Laboratories Inc.
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