Alpha Analytical, Inc.
Facility: Mansfield
Department: Organic Extractions
Title: Percent Lipids Determination
ID No.:2171
Revision 5
Published Date:3/12/2013 11:38:20 AM
Page 1 of 8
Percent Lipids Determination
References: NOAA Technical Memorandum NOS ORCA 130: Sampling and Analytical
Methods of the National Status and Trends Program Mussel Watch Project:
1993-1996 Update. March 1998.
1.
Scope and Application
Matrices: Tissue sample extractions.
Definitions: Refer to Alpha Analytical Quality Manual.
This Standard Operating Procedure (SOP) describes the percent lipid determination as adopted by
Alpha Analytical. Results derived from this method have been compared, with remarkable accuracy,
to nationally published results for both fish and mussel tissue extracts.
This method has been developed to determine the percent lipid or total extractable organic (TEO) of
any tissue sample. Data derived from this method may be used to assist toxicologists when weighing
human health or ecological risk assessments.
The data report packages present the documentation of any method modification related to the
samples tested. Depending upon the nature of the modification and the extent of intended use, the
laboratory may be required to demonstrate that the modifications will produce equivalent results for
the matrix. Approval of all method modifications is by one or more of the following laboratory
personnel before performing the modification: Area Supervisor, Department Supervisor, Laboratory
Director, or Quality Assurance Officer.
This method is restricted to use by or under the supervision of experienced analysts. Each analyst
must demonstrate the ability to generate acceptable results with this method by performing an initial
demonstration of capability, analyzing a proficiency test sample and completing the record of
training.
After initial demonstration, ongoing demonstration is based on acceptable laboratory performance of
at least a quarterly laboratory control sample or acceptable performance from an annual proficiency
test sample. A major modification to this procedure requires demonstration of performance. The
identification of major method modification requiring performance demonstration is directed by the
Quality Assurance Officer and/or Laboratory Director on a case-by-case basis.
2.
Summary of Method
An aliquot of tissue sample extract is taken, prior to any sample cleanup process;.the aliquot is
weighed and the extraction solvent is allowed to evaporate overnight under a fume hood. The
aliquot is then re-weighed. Calculations are applied (Section 11) to determine the percent lipid
content.
If more than one method for organic analysis is being performed, the laboratory Department
Manager will select which tissue extract is to be used for percent lipid determination. It is
recommended that the method with the largest final volume be chosen.
2.1 Method Modifications from Reference
The aliquot of tissue extract was reduced from 20mL to 250-500uL. This eliminates the need to
re-concentrate the samples for lipid determination. The intermediate steps of reducing the
sample aliquot to total dryness under nitrogen, re-wetting, and re-mixing the sample by adding
Printouts of this document may be out of date and should be considered uncontrolled. To accomplish work,
the published version of the document should be viewed online.
Document Type: SOP-Technical
Pre-Qualtrax Document ID: OP-015
Alpha Analytical, Inc.
Facility: Mansfield
Department: Organic Extractions
Title: Percent Lipids Determination
ID No.:2171
Revision 5
Published Date:3/12/2013 11:38:20 AM
Page 2 of 8
1mL of DCM, then removing 100uL of sample extract for re-drying for the final lipid weight are
replaced by the steps in Section 10.3.3 and 10.3.4. The evaluation of SRMs with known lipid
values has proven over time the accuracy of this procedure.
3.
Reporting Limits
The reporting limit is 0.01% and is largely based on the tolerance of the balance used for the
determination.
4.
Interferences
4.1 It is important that all weighing be performed at ambient temperatures as hot or chilled
items will lose or gain weight with temperature fluctuations.
4.2 The percent lipid determination must be performed on the extract before any cleanup
procedures are performed
4.3 All weights must be recorded to four decimal places using an Analytical balance. Small
differences in weights can significantly affect the final calculation.
5.
Health and Safety
The toxicity or carcinogenicity of each reagent and standard used in this method is not fully
established; however, each chemical compound should be treated as a potential health hazard.
From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by
whatever means available. A reference file of material safety data sheets is available to all personnel
involved in the chemical analysis. Additional references to laboratory safety are available in the
Chemical Hygiene Plan.
All personnel handling environmental samples known to contain or to have been in contact with
municipal waste must follow safety practices for handling known disease causative agents.
The use of laboratory equipment and chemicals exposes the analyst to several potential hazards.
Good laboratory techniques and safety practices shall be followed at all times. All relevant Material
Safety Data Sheets (MSDSs) are kept on file.
Approved PPE, which includes Safety Glasses, Gloves and Lab Coats, must be worn at all times
when handling samples, reagents, chemicals, or when in the vicinity of others handling these items,
so that dermal contact is avoided. All standards, reagents and solvents shall be handled under a
hood. All flammable solvents must be kept in the flammable storage cabinet, and returned to the
cabinet immediately after use. When transporting chemicals, use a secure transporting devise
and/or secondary outer container. Chemical storage is properly segregated and adequately
ventilated to reduce the possibility of hazardous reactions. Chemical storage in work areas shall be
kept to a minimum. Storage on bench tops or other work surfaces, except temporary, is not
permitted.
All standards and reagents shall be prepared in a hood while using the proper PPE.
All additional company safety practices shall be followed at all times as written the Chemical Hygiene
Plan.
6.
Sample Collection, Preservation, Shipping and Handling
6.1 Sample Collection
Not applicable.
Printouts of this document may be out of date and should be considered uncontrolled. To accomplish work,
the published version of the document should be viewed online.
Document Type: SOP-Technical
Pre-Qualtrax Document ID: OP-015
Alpha Analytical, Inc.
Facility: Mansfield
Department: Organic Extractions
Title: Percent Lipids Determination
ID No.:2171
Revision 5
Published Date:3/12/2013 11:38:20 AM
Page 3 of 8
6.2 Sample Preservation
Samples and resulting extracts are stored at -20°C + 10°C. No other preservatives are used.
6.3 Sample Shipping
No special sample shipping requirements.
6.4 Sample Handling
Samples and resulting extracts shall be stored at -20°C + 10°C. Hold times are not applicable
to the percent lipid determination.
7.
Equipment and Supplies
7.1 Analytical balance: Capable of weighing to the nearest 0.0001g. The Cahn
microbalance is also suitable.
7.2 Aluminum weighing tins
7.3 Fume hood
7.4 Gas-tight Syringes: Various volumes, solvent rinsed with methylene chloride
8.
Reagents and Standards
8.1 Sodium sulfate: Granular, anhydrous. Purified by heating to 400°C for 4 hours prior to
use.
8.2 Glass wool: Purified by heating to 400°C for 1 hour prior to use.
8.3 Methylene chloride: ACS approved, Pesticide grade.
9.
Quality Control
The laboratory must maintain records to document the quality of data that is generated. Ongoing
data quality checks are compared with established performance criteria to determine if the results
of analyses meet the performance characteristics of the method.
9.1 Blank
A method blank must be analyzed with every batch. This blank consists of an aluminum
weighing tin, with no sample extract added. The result of the method blank must be less than
0.01%.
Corrective Action: If the blank result is >0.01%, recalculate the results. If the blank result is still
> 0.01%, repeat the tissue extraction for all associated samples and reset the percent lipid
determination on the re-extracts. Note: Depending upon the amount of original tissue sample
remaining, repeat extractions may not be possible.
9.2 Laboratory Control Sample (LCS)
Not applicable to this method.
Printouts of this document may be out of date and should be considered uncontrolled. To accomplish work,
the published version of the document should be viewed online.
Document Type: SOP-Technical
Pre-Qualtrax Document ID: OP-015
Alpha Analytical, Inc.
Facility: Mansfield
Department: Organic Extractions
Title: Percent Lipids Determination
ID No.:2171
Revision 5
Published Date:3/12/2013 11:38:20 AM
Page 4 of 8
9.3 Initial Calibration Verification (ICV)
Not applicable to this method.
9.4 Continuing Calibration Verification (CCV)
Not applicable to this method.
9.5 Matrix Spike
Not applicable to this method.
9.6 Laboratory Duplicate
A Duplicate analysis is performed once per 20 samples, or at least 1 per batch, whichever is
more frequent. The RPD between the duplicates must be less than 20%
Corrective Action: If the RPD between the duplicates exceeds 20% the analyst shall check the
calculations for errors. If no errors are found, repeat the tissue extraction for the batch. Then
re-perform the percent lipid determination on the re-extracts. Note: Depending upon the
amount of original tissue sample remaining, repeat extractions may not be possible.
9.7 Method-specific Quality Control Samples
None.
9.8 Method Sequence
• Method Blank
• Laboratory Duplicate
• Samples 1-19
10.
Procedure
10.1
Equipment Set-up
The Analytical balance, or Cahn balance, used to make each weighing for this method is
checked daily before use with Class 1 weights, in the weight range of use, by a designated
analyst, or appointed alternate. These daily checks are documented in the “Balance Calibration
Check” logbook. The balance is serviced semiannually by an outside service technician.
10.2
Initial Calibration
Not applicable to this method.
10.3
Equipment Operation and Sample Processing
10.3.1 Samples are extracted per the Microscale Solvent Extraction SOP (OP-016) or the
Tissue Extraction SOP (OP-018)
10.3.2 Label, weigh and record the weight of an aluminum weighing tin on the Excel spread
sheet (see Figure 1), under “weight, t”. Note: Record ALL weights to four decimal places
(nearest 0.0001g). NOTE: Record name, date and time as well on Excel sheet
10.3.3 Using a methylene chloride-rinsed 250ul-500ul gas-tight syringe, remove 250ul-500ul of
the dried sample extract and gently expel the 250ul-500ul extract into the pre-weighed
and labeled aluminum weighing tin from Section 10.3.2, above. Record the volume of the
aliquot on the Excel spread sheet under “Sample Aliquot”.
10.3.4 Place the tin under a fume hood overnight to allow for complete solvent evaporation.
Printouts of this document may be out of date and should be considered uncontrolled. To accomplish work,
the published version of the document should be viewed online.
Document Type: SOP-Technical
Pre-Qualtrax Document ID: OP-015
Alpha Analytical, Inc.
Facility: Mansfield
Department: Organic Extractions
Title: Percent Lipids Determination
ID No.:2171
Revision 5
Published Date:3/12/2013 11:38:20 AM
Page 5 of 8
10.3.5 Re-weigh the tin with the dried extract the following day, and record the weight on the
Excel spreadsheet under “weight, g1”.
10.4
Continuing Calibration
Not applicable for this method.
10.5
Preventive Maintenance
10.5.1 Analytical and Cahn Balance
11.
10.5.1.1
All balances are calibrated and serviced every six months by an instrument
service company. All service records are kept on file.
10.5.1.2
Keep balances clean.
Data Evaluation, Calculations and Reporting
11.1 Calculate the Percent Lipids as follows, an example spreadsheet is shown in Figure 1:
(W d + a) – W d = Net weight, g
(FV / Va) x Net weight x 1000 = TEW
TEW / W ext = TEC
TEC / 10 = % Lipid
where:
Wd + a =
Weight of the weighing tin and the extract, in grams, as recorded in
Section 10.3.6.
Wd =
Weight of the weighing tin, in grams, as recorded in Section 10.3.2.
W ext =
Weight of tissue extracted, in grams, wet, (from extraction logbook).
FV =
Final volume of the extract, in microliters, (i.e., 58000uL = 58.0mL).
Va =
Volume of the extract aliquot recorded in Section 10.3.3, in microliters
(i.e., 5000uL = 5.0mL).
TEW =
Total Extractable Weight
TEC =
Total Extractable Concentration
11.2 Procedures for data and record management must adhere to the Quality Systems Manual,
other subordinate documents covering record keeping, and the Document Control SOP,
(SOP/08-01). All records shall be stored in such a manner as to be safe and accessible for at
least 10 years.
11.3 Notebooks: Laboratory notebooks are designed to accommodate the specific analysis. If a
sample requires re-analysis or re-extraction for any reason, a notation is made next to the
sample entry in that log. Such logbooks shall be archived so as to remain available for at least
10 years. All laboratory notebooks must follow the specifications in the Laboratory Notebook
Usage Work Instruction, 108-01, and follow all record keeping and document control practices.
Printouts of this document may be out of date and should be considered uncontrolled. To accomplish work,
the published version of the document should be viewed online.
Document Type: SOP-Technical
Pre-Qualtrax Document ID: OP-015
Alpha Analytical, Inc.
Facility: Mansfield
Department: Organic Extractions
Title: Percent Lipids Determination
ID No.:2171
Revision 5
Published Date:3/12/2013 11:38:20 AM
Page 6 of 8
11.4 To report data, go to the LIMS and “batch” and “associate” all samples in this analytical batch.
11.5 Enter the results obtained from the Excel spreadsheet.
11.6 Forward the hand recorded raw data from the Excel spreadsheet and the LIMS batch printout
to the Department Manager for secondary review.
11.7 All results are reportable without qualification if the QC criteria defined in Section 9 are met. If
any QC parameters are not met, all associated samples must be evaluated for re-extraction
and/or re-analysis. See section 9 for discussion, including corrective action.
12.
Contingencies for Handling Out-of-Control Data or Unacceptable
Data
Corrective Action for non-compliant data is discussed in Section 9. If a situation arises (such as loss
of sample, or the inability for re-extraction or re-analysis) that requires additional action, the Project
Manager and/or Laboratory Director are immediately notified and the client is contacted for
discussion and/or instruction for any further action. The situation and the agreed upon actions will
be discussed in the project narrative that accompanies the data package.
13.
Method Performance
13.1
Method Detection Limit Study (MDL) / Limit of Detection Study (LOD) /
Limit of Quantitation (LOQ)
The laboratory follows the procedure to determine the MDL, LOD, and/or LOQ as outlined in
Alpha SOP/08-05. These studies performed by the laboratory are maintained on file for review.
13.2 Demonstration of Capability Studies
Refer to Alpha SOP/08-12 for further information regarding IDC/DOC Generation.
13.2.1
Initial (IDC)
The analyst must make an initial, one-time, demonstration of the ability to generate
acceptable accuracy and precision with this method, prior to the processing of any
samples.
13.2.2
Continuing (DOC)
The analyst must make a continuing, annual, demonstration of the ability to generate
acceptable accuracy and precision with this method.
14.
Pollution Prevention and Waste Management
All solvent waste generated during lipid analysis must be stored in satellite containers in the
preparation laboratory labeled “Organic Solvent”.
Once the organic solvent satellite containers are full, they must be emptied into a 55-gallon drum
marked “Organic Solvent Waste”.
Refer to Alpha’s Chemical Hygiene Plan and Waste Management and Disposal SOP for further
pollution prevention and waste management information.
Printouts of this document may be out of date and should be considered uncontrolled. To accomplish work,
the published version of the document should be viewed online.
Document Type: SOP-Technical
Pre-Qualtrax Document ID: OP-015
Alpha Analytical, Inc.
Facility: Mansfield
Department: Organic Extractions
Title: Percent Lipids Determination
15.
ID No.:2171
Revision 5
Published Date:3/12/2013 11:38:20 AM
Page 7 of 8
Referenced Documents
SOP/OP-004 Extraction of Soil, Tissue, Vegetation and Sediment Samples by Pressurized Fluid
Extraction
WI/108-01 Laboratory Logbook Usage
Chemical Hygiene Plan
SOP/01-01 Sample Receipt and Login
SOP/01-02 Sample Custody and Tracking
SOP/08-01 Document Control
SOP/08-05 MDL/LOD/LOQ Generation
SOP/08-12 IDC/DOC Generation
SOP/G-006 Waste Management and Disposal SOP
16.
Attachments
Figure1: Example Excel Spreadsheet
Printouts of this document may be out of date and should be considered uncontrolled. To accomplish work,
the published version of the document should be viewed online.
Document Type: SOP-Technical
Pre-Qualtrax Document ID: OP-015
Alpha Analytical, Inc.
Facility: Mansfield
Department: Organic Extractions
Title: Percent Lipids Determination
ID No.:2171
Revision 5
Published Date:3/12/2013 11:38:20 AM
Page 8 of 8
Figure 1
Example Excel Spreadsheet
Method #: LIPIDS
Set up Date: 8/20/02
Analyst: TLB
QC
Sample
ETR#
No.
BLANKTINB
207077 16
Sample Lipid Final Sample Extract
Dish Weight Dilution Volume Aliquot Aliquot
D No. (wet) Factor (mL) (uL)
(uL)
B
16
5.00
5.42
1.09
1.09
59
60
5000
5000
0.00
5000
0.00
5000
0.00
5000
0.00
5000
0.00
5000
0.00
5000
0.00
5000
0.00
5000
(g)
weight
g1 0.9565
59000 t 0.9565
n1 0.0000
g1 0.9710
60000 t 0.9567
n1 0.0143
g1
0
t
n1 0.0000
g1
0
t
n1 0.0000
g1
0
t
n1 0.0000
g1
0
t
n1 0.0000
g1
0
t
n1 0.0000
g1
0
t
n1 0.0000
g1
0
t
n1 0.0000
g1
0
t
n1 0.0000
TOTAL
TOTAL
EXTRACTABLE
EXTRACTABLE
WEIGHT
CONC.
%
LIPIDS n=net solid
Calculations
t=tare wt.
g=gross solid wt.
calculations:
0.00
0.00
0.000
n=g-t
TOTAL Ext Wt =((ext. vol in uL)/(aliquot vol in uL))*(n1*1000)
TOTAL Ext Conc = (Total Ext Wt)/Sample Wt
171.60
31.66
3.166
0.00
#DIV/0! #DIV/0!
0.00
#DIV/0! #DIV/0!
0.00
#DIV/0! #DIV/0!
0.00
#DIV/0! #DIV/0!
0.00
#DIV/0! #DIV/0!
0.00
#DIV/0! #DIV/0!
0.00
#DIV/0! #DIV/0!
0.00
#DIV/0! #DIV/0!
%Lipids = Total Ext. Conc. / 10
TEMPLATE: lipid.XLT
Printouts of this document may be out of date and should be considered uncontrolled. To accomplish work,
the published version of the document should be viewed online.
Document Type: SOP-Technical
Pre-Qualtrax Document ID: OP-015