EPA 600/R-13/022 | August 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
CENTERS FOR DISEASE"
CONTROL AND PREVENTION
High Throughput Determination
of Ricinine, Abrine, and
Alpha-Amanitin in Drinking
Water by Solid Phase Extraction
and High Performance Liquid
Chromatography Tandem Mass
Spectrometry (HPLC/MS/MS)
Version 1.0
Office of Research and Development
National Homeland Security Research Center
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EPA/600-R-13/022
August 2013
High Throughput Determination of Ricinine, Abrine, and Alpha-Amanitin in Drinking
Water by Solid Phase Extraction and High Performance Liquid Chromatography Tandem
Mass Spectrometry (HPLC/MS/MS)
Version 1.0
Centers for Disease Control and Prevention
Atlanta, GA 30333
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and
Development funded and managed the research described herein under EPA IA# DW75-
92259701 with the Centers for Disease Control and Prevention. This content has been peer and
administratively reviewed and has been approved for publication as a joint EPA and CDC
document. Note that approval does not signify that the contents necessarily reflect the views of
the EPA, the CDC, the Public Health Service, or the U.S. Department of Health and Human
Services. Reference herein to any specific commercial product, process, or service by trade
name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its
endorsement, recommendation, or favoring by the United States government. The views and
opinions expressed herein do not necessarily state or reflect those of the United States
government and shall not be used for advertising or product endorsement purposes.
Questions concerning this document or its application should be addressed to:
Erin Silvestri, MPH (EPA Project Officer)
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
513-569-7619
Silvestri.Erin@epa.gov
Matthew Magnuson, PhD (EPA Technical Lead)
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
513-569-7321
Magnuson.Matthew@epa. gov
Jennifer Knaack, PhD
Mercer University
3001 Mercer University Drive
Atlanta, GA 30341
678-547-6737
knaack J s@mercer. edu
Rudolph Johnson, PhD
Centers for Disease Control and Prevention
4770 Buford Highway, MS F-44
Atlanta, GA 30341
770-488-3543
Rmj6@cdc.gov
ii
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Acknowledgments
The following researchers were critical to the development of the method and preparation of the
procedure:
Centers for Disease Control and Prevention, National Center for Environmental Health
Jennifer Knaack (currently at Mercer University)
Rudolph Johnson
Christopher Pittman
Joe Wooten
The following individuals served as members and technical advisors of the Project Team:
U.S. Environmental Protection Agency (EPA), Office of Research and Development,
National Homeland Security Research Center
Matthew Magnuson (EPA Technical Lead)
Erin Silvestri (EPA Project Officer)
Sanjiv Shah
in
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Table of Contents
Disclaimer ii
Acknowledgments iii
List of Tables and Figures iv
List of Acronyms v
Executive Summary vi
1. SCOPE AND APPLICATION 1
2. SUMMARY OF METHOD 3
3. DEFINITIONS 3
4. INTERFERENCES 5
5. SAFETY 6
6. EQUIPMENT AND SUPPLIES 6
7. REAGENTS AND STANDARDS 10
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE 15
9. QUALITY CONTROL 18
10. CALIBRATION AND STANDARDIZATION 25
11. PROCEDURE 29
12. DATA ANALYSIS AND CALCULATIONS 32
13. METHOD PERFORMANCE 33
14. POLLUTION PREVENTION 35
15. WASTE MANAGEMENT 35
16. REFERENCES 36
List of Tables and Figures
Table 6-1. High Performance Liquid Chromatography (HPLC) Parameters 8
Table 6-2. Tandem Mass Spectrometer (MS/MS) Parameters 9
Table 6-3. Mass Spectrometer conditions 9
Table 7-1. Calibration Standard Stock Solution Volumes 14
Table 8-1. Preservative Concentrations and Purposes of Preservatives 16
Table 8-2. Recoveries of Ricinine, Abrine, and Alpha-Amanitin in Preservative Over Time ....17
Table 10-1. Ion Transitions Monitored for Ricinine, Abrine, and Alpha-Amanitin 26
Table 13-1. Method Performance 34
Table 13-2. Single Laboratory Precision and Accuracy Data 34
Figure 6-1. Chromatogram of typical analyte and internal standard peaks 10
iv
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Acronyms
ABR abrine
AMAN alpha-amanitin
CAS® Chemical Abstracts Service
CCC continuing calibration check
CDC Centers for Disease Control and Prevention
CR confirmation ratio
DL detection limit
ESI electrospray ionization
FD field duplicate
HRpir half range for the predicted interval of results
HRL highest reportable limit
HPLC high performance liquid chromatography
i.d inside diameter
IDC initial demonstration of capability
IS internal standard
IQS internal quantification standard
LC liquid chromatography
LDso median lethal dose
LFB laboratory fortified blank
LFSM laboratory fortified sample matrix
LFSMD laboratory fortified sample matrix duplicate
LRB laboratory reagent blank
MRL minimum reporting level
MRM multiple reaction monitoring
MS/MS tandem mass spectrometry
MSDS Material Safety Data Sheet
m/z mass to charge ratio
NHSRC National Homeland Security Research Center
PIR prediction interval of results
QC quality control
QCS quality control sample
RIC ricinine
RPD relative percent difference
SAP Sampling and analytical procedure
SPE solid phase extraction
SS standard solution
TOC Total Organic Carbon
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Executive Summary
This document provides the standard operating procedure for determination of ricinine (RIC),
abrine (ABR), and a-amanitin (AMAN) in drinking water by isotope dilution liquid
chromatography tandem mass spectrometry (LC/MS/MS). This method is designed to support
site characterization and inform site-specific cleanup goals of environmental remediation
activities following a homeland security incident involving one or a combination of these
analytes.
The method can be summarized as follows. A 50-mL drinking water sample is collected and
preserved with sodium thiosulfate (80 mg/L) (a dechlorinating agent), and sodium Omadine™
(64 mg/L) (an antimicrobial preservative). An aliquot is combined with an internal standard
mixture containing isotopically-labeled standards (for RIC and ABR) and/or internal
quantification standard (for AMAN). The sample is then pipetted into a well of a preconditioned
96-well solid phase extraction plate and extracted. The extract is concentrated to dryness under
nitrogen and heat and is then adjusted to a 100 uL volume in high performance liquid
chromatography (HPLC)-grade water. RIC, ABR, and AMAN are separated from the sample
matrix and identified by HPLC/MS/MS analysis, operated in multiple reaction monitoring mode
or equivalent. Analyte identification is accomplished by comparing the acquired mass spectra,
including ion ratios and retention times, to reference spectra and retention times for calibration
standards acquired under identical HPLC/MS/MS conditions. Quantitation is performed using
the internal standard technique. Utilization of an isotopically labeled internal standard for RIC
and ABR and an internal quantification standard for AMAN provides a high degree of accuracy
and precision for sample quantitation by accounting for analyte recovery and analytical
efficiency.
Accuracy and precision data have been generated in reagent water and in finished ground and
surface waters that contain residual chlorine and/or chloramine that have been used as
disinfectants. RIC, ABR, and AMAN can be analyzed up to 28 days after collection, although
initial loss of analyte signal during the first few hours may be observed with ABR and AMAN in
some water samples. The amount of loss is dependent on the water source and does not continue
after this initial period. Thus, in an actual contamination incident, it is likely this initial signal
loss would occur before the sample reaches the laboratory.
VI
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HIGH THROUGHPUT DETERMINATION OF RICININE, ABRINE, and ALPHA-
AMANITIN IN DRINKING WATER
1. SCOPE AND APPLICATION
1.1. This document is an isotope dilution liquid chromatography tandem mass
spectrometry (LC/MS/MS) method for the determination of ricinine (1,2-dihydro-
4-methoxy-l-methyl-2-oxo-3-pyridinecarbonitrile, Chemical Abstracts Services
(CAS®) Registry Number® 524-40-3), abrine ((2S)-3-(lH-indol-3-yl)-2-
(methylamino)propanoic acid, CAS Registry Number 526-31-8), and alpha-
amanitin (cyclic(L-asparaginyl-4-hydroxy-L-prolyl-(R)-4,5-dihydroxy-L-
isoleucyl-6-hydroxy-2-mercapto-L-tryptophylglycyl-L-isoleucylglycyl-L-
cysteinyl), cyclic (4-8)-sulfide, (R)-S-oxide, CAS Registry Number 23109-05-9).
This method, including quality control (QC) requirements, is designed to support
site characterization and to inform site-specific cleanup goals of environmental
remediation activities following a homeland security incident involving one or a
combination of these analytes. (Note: Ricin, abrin, and a-amanitin are not toxins
regulated under the Safe Drinking Water Act (as amended in 1986 and 1996) [1].
A description of the development of this method will be presented separately.
1.1.1. Ricinine (RIC), abrine (ABR), and alpha-amanitin (AMAN) can be
analyzed up to 28 days after collection. However, in some water samples,
ABR and AMAN were observed to undergo an initial loss of analyte
signal during the first few hours after ABR and AMAN were added to the
sample (Section 8.4). The amount of loss is dependent on the water
source, does not continue after this initial period, and will be discussed in
more detail elsewhere [2]. Thus, in an actual contamination incident, it is
likely this initial signal loss would occur before the sample reaches the
laboratory. Section 13.5 provides suggestions for additional quality
control steps that may be necessary should analysis of samples suspected
of ABR or AMAN contamination results in unexpectedly low reported
concentrations.
1.2. Significance: Ricin and abrin are toxic lectins from Ricinus communis (castor
bean) [3,4] and Abrusprecatorias (jequirity pea) [5,6], respectively. Ricinine and
abrine are small alkaloid molecules that are present in crude extracts of ricin and
abrin, respectively, and can be used as biomarkers for these toxins [7-9]. Alpha-
Amanitin is a highly toxic bicyclic octapeptide found in Amanitaphalloides
[10,11]. Methods for detecting these toxins in environmental matrices are
necessary to characterize the contamination and to inform remediation and
clearance of intentional or accidental contamination events. The method presented
here can be applied for the quantification of ricinine, abrine, and alpha-amanitin
in drinking water samples.
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1.3. Whether performed manually or with automation, the use of 96-well plates during
solid phase extraction (SPE) produces two key benefits. The 96-well plates allow
for extensive automation of the method, thereby enabling high throughput of
samples, as might be required during environmental remediation. Additionally,
the use of this format results in the ability to perform isotope dilution more
economically since smaller amounts can be utilized.
1.4. Isotopically-labeled RIC and ABR internal standards and an internal
quantification standard (IQS) for AMAN are added equally to all unknowns,
quality controls, and calibration standards. In addition to enabling accurate
quantitation, internal standards also account for and resolve some of the QC
issues surrounding analysis, including analysis efficiency and sample loss, in the
intended use of these analytes. The overall QC approach utilizing quantitation and
confirmation ions as well as either an isotopically-labeled analyte or internal
quantification standard (see Section 3.9) greatly increases confidence that RIC,
ABR, and AMAN, and not another molecule with similar fragmentation patterns,
are being quantitated during analysis.
1.5. For this method, accuracy and precision data have been generated in reagent
water and in finished ground and surface waters that contain residual chlorine
and/or chloramine that have been used as disinfectants.
1.6. The QC approach included in this method has been single-laboratory verified by
the Centers for Disease Control and Prevention (CDC), National Center for
Environmental Health, Division of Laboratory Sciences, Emergency Response
Branch, in the Chemical Terrorism Methods Development Laboratory and is
designed to be sufficiently rigorous that network laboratories can successfully
perform the method.
1.7. The minimum reporting level (MRL) is the lowest analyte concentration that
meets data quality objectives for the intended use of the method, e.g., to meet site-
specific remediation goals. Laboratories will need to demonstrate that their
laboratory MRL meets the requirements described in Section 9.2.4.
1.8. Determining the detection limit (DL) is optional (Section 9.2.6). Detection limit is
defined as the statistically calculated minimum concentration that can be
measured with 99% confidence that the reported value is greater than zero.
1.9. This method is intended for use by analysts skilled in the performance of solid
phase extractions, the operation of high performance liquid
chromatography/tandem mass spectrometry) HPLC/MS/MS instruments and the
interpretation of the associated data.
1.10. This method has been verified using only the conditions specified in the method.
Alteration of this method is not recommended. However, equivalent equipment
and consumables are acceptable when equivalent performance is demonstrated.
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2. SUMMARY OF METHOD
2.1. A 50-mL water sample is collected and preserved with sodium thiosulfate (80
mg/L) (a dechlorinating agent), and sodium Omadine™ (64 mg/L) (an
antimicrobial preservative, Arch Chemicals, Inc., Norwalk, CT). An aliquot is
combined with an internal standard mixture containing isotopically-labeled
standards (for RIC and ABR) and/or internal quantification standard (for AMAN).
The sample is then pipetted into a well of a preconditioned 96-well solid phase
extraction plate and extracted. The extract is concentrated to dryness under
nitrogen and heat and is then adjusted to a 100 uL volume in HPLC-grade water.
RIC, ABR, and AMAN are separated from the sample matrix and identified by
HPLC/MS/MS analysis, operated in multiple reaction monitoring (MRM) mode
or equivalent. Analyte identification is accomplished by comparing the acquired
mass spectra, including ion ratios and retention times, to reference spectra and
retention times for calibration standards acquired under identical HPLC/MS/MS
conditions. Quantitation is performed using the internal standard technique.
Utilization of an isotopically labeled internal standard for RIC and ABR and an
internal quantification standard for AMAN provides a high degree of accuracy
and precision for sample quantitation by accounting for analyte recovery and
analytical efficiency.
2.2. Due to site-specific circumstances during an environmental remediation activity,
changes to the on-going calibration frequency, and number of continuing
calibration checks (CCCs) may be necessary and appropriate. For example, this
may be necessary when sample throughput requirements exceed available
laboratory capacity or when a shorter initial demonstration of capability (IDC) is
warrented. However, initial and ongoing QC requirements and acceptance criteria
(see Section 9) should not be changed. Adopting steps such as a replacing
ongoing recalibration with a calibration check only to save time may result in
higher QC failure rates and perhaps less accurate quantitation. Laboratories
should discuss these increased risks with sample submitters before taking such
steps.
3. DEFINITIONS
3.1. ANALYSIS BATCH - a sequence of samples analyzed within a 24-hour period,
including no more than 20 field samples in addition to all of the required QC
samples (Section 9.3).
3.2. CALIBRATION STANDARD STOCK SOLUTION - A solution prepared from
the primary dilution standard solution(s) and/or stock standard solution(s) and the
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internal standard(s). The calibration standard stock solutions are used to calibrate
the instrument response with respect to analyte concentration.
3.3. CONFIRMATION ION TRANSITION - for this method, the confirmation ion
transition is the second most abundant ion transition for each analyte (See
Confirmation Ratio, Section 3.4, below).
3.4. CONFIRMATION RATIO (CR) - the peak area produced by the confirmation
ion transition divided by the peak area produced by the quantitation ion transition.
The confirmation ratio serves as an additional QC measure for analyte
identification.
3.5. CONTINUING CALIBRATION CHECK (CCC) SOLUTION - a calibration
solution containing the method analyte(s), which is extracted in the same manner
as the samples and analyzed periodically to verify the accuracy of the existing
calibration for those analyte(s).
3.6. DETECTION LIMIT (DL) - the minimum concentration of an analyte that can be
identified, measured, and reported to be greater than zero with 99% confidence.
3.7. FIELD DUPLICATES (FD1 and FD2) - two separate samples collected at the
same time and place under identical circumstances and treated exactly the same
throughout field and laboratory procedures to check the precision associated with
sample collection, preservation, storage, and laboratory procedures.
3.8. ISOTOPICALLY-LABELED INTERNAL STANDARD (IS) - a pure chemical
added to an extract or to a standard solution in a known amount(s) and used to
measure the relative response of other method analytes and surrogates that are
components of the same solution.
3.9. LABORATORY FORTIFIED BLANK (LFB) - a volume of reagent water or
other blank matrix to which known quantities of the method analytes and all the
preservation reagents are added in the laboratory (Section?.3.5.2). The LFB is
analyzed exactly like a sample. Its purpose is to verify that the methodology is
competently replicated and that the laboratory is capable of making accurate and
precise measurements.
3.10. LABORATORY REAGENT BLANK (LRB) - an aliquot of reagent water that is
treated exactly as a sample and used to determine if method analytes or other
interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.11. MATERIAL SAFETY DATA SHEET (MSDS) - written information provided
by vendors detailing a chemical's toxicity, health hazards, physical properties, fire
and reactivity data, and precautions for storage, spill, and handling.
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3.12. MINIMUM REPORTING LEVEL (MRL) - the minimum concentration qualified
to be reported as a quantitated value for a method analyte in a sample following
analysis (Section 9.2.4. for MRL verification procedure).
3.13. PRIMARY DILUTION STANDARD SOLUTION - a solution containing the
analytes prepared in the laboratory from stock standard solutions and diluted as
needed to prepare calibration solutions and other needed analyte solutions.
3.14. QUANTITATION ION TRANSITION -for this method, the quantitation ion
transition is the most abundant ion transition for each analyte and internal
standard (See Confirmation Ratio, Section 3.4, above). Only quantitation ion
transitions are monitored for internal standards.
3.15. SECOND SOURCE QUALITY CONTROL SAMPLES - materials obtained
from a source different from the original source and used to verify the accuracy of
the existing calibration for those analytes.
4. INTERFERENCES
4.1. Method interferences that can lead to discrete artifacts and/or elevated baselines
in the chromatograms may be caused by contaminants in solvents, reagents
(including reagent water), sample bottles and caps, and other sample processing
hardware. All such items must be routinely demonstrated to be free from
interferences under the conditions of the analysis by analyzing laboratory reagent
blanks. Subtracting blank values from sample results is not permitted.
4.2. Matrix interferences may be caused by contaminants that are co-extracted from
the sample. The extent of matrix interferences will vary considerably from source
to source, depending upon the nature of the water.
4.3. Relatively high concentrations, in the mg/L range, of preservatives, antimicrobial
agents, or dechlorinating agents might be added to sample collection vessels
(Section 8.1.2). The potential exists for trace-level organic contaminants in these
reagents. Interferences from these sources should be monitored by analysis of
laboratory reagent blanks particularly when new lots of reagents are acquired.
4.4. Due to the nature of the matrix analyzed in this procedure, occasional
interferences from unknown substances in the reagents and supplies used in this
method might be encountered. Interfering compounds can be recognized by
deviations in the sample quantitation/confirmation ratios from the calibration
standard ratios and can also be monitored using appropriate LRBs. Any
interference that results in quality control (QC) failure (Section 9) results in
rejection of the entire analysis batch. If repeating the analysis does not remove the
interference with the reference standard, the results for that analyte are not
reportable.
4.5. All glassware should be chemically cleaned before running this method. Wash
glassware thoroughly with reagent-grade water followed by acetonitrile. Allow
glass to dry completely before use. If the laboratory wishes to use a muffle oven
for decontamination, then the appropriate measures should be taken to assure that
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the muffle oven conditions are suitable to remove all traces of RIC, ABR, AMAN
and other interferences.
4.6. Care should be taken at all times to prevent contamination of QC materials,
standards, and samples.
4.7. Chromatographic separation of the analyte should be carefully monitored for
unknown interferences. See Section 11.2.5 for analyte confirmation.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been
defined precisely. Each chemical should be treated as a potential health hazard,
and exposure to these chemicals should be minimized. Each laboratory is
responsible for maintaining an awareness of Occupational Safety and Health
Administration regulations regarding safe handling of chemicals used in this
method. A reference file of MSDSs should be made available to all personnel
involved in the chemical analyses. Additional references to laboratory safety are
available [12-14].
5.2 While this method determines ricinine and abrine, ricin and abrin may also be
present in the samples. Ricin is toxic, and the rat oral LDso has been reported
between 1-20 mg toxin/kg body weight [15,16]. Abrin toxicity in beverages is
reported to be within the same range in mice [17]. Alpha-Amanitin is also highly
toxic and the LD50 due to ingestion is 0.1-0.3 mg toxin/kg body weight [10,11].
Ingestion is the primary reported route of exposure, but all other routes of
exposure (e.g., inhalation, dermal contact, and eye exposure) should be avoided.
Follow universal safety precautions when performing this procedure, including
the use of a laboratory coat, safety glasses, latex or nitrile gloves, and a high
quality ventilated chemical fume hood and/or biological safety cabinet.
5.3 Avoid inhalation or dermal exposure to acetonitrile, methanol, and formic acid,
which are used in the sample preparation and analysis steps.
5.4 Mechanical hazards when performing this procedure using standard safety
practices are minimal. Read and follow the manufacturers' information regarding
safe operation of the equipment. Avoid direct contact with the mechanical and
electronic components of the liquid chromatograph and mass spectrometer unless
all power to the instrument is off. Maintenance and repair of mechanical and
electronic components should generally be performed only by qualified
technicians.
6. EQUIPMENT AND SUPPLIES (It is important to note that specific brands or catalog
numbers included in this section are examples only and do not imply endorsement of
these particular products. These specific products were used during the validation of this
method.)
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6.1 MICRODISPENSERS - with adjustable volume (5-100 uL, 100-1000 uL)
(Eppendorf Co., Westbury, NY, or equivalent).
6.2 REPEATER® PIPETTE - Eppendorf 4780 (Eppendorf Co., Westbury, NY, or
equivalent).
6.3 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g.
6.4 SOLID PHASE EXTRACTION (SPE) APPARATUS WITH 96 WELL PLATES
6.4.1 96-WELL PLATE SPE- Strata™ X 60-mg 96-well plate (PN# 8E-S100-
UGB), available from Phenomenex (Torrence, CA) or equivalent.
6.4.2 PLATE SHAKER (ThermoFisher Scientific, Waltham, MA, or
equivalent).
6.4.3 96-WELL LIQUID HANDLER - Use a 96-well liquid handler equipped
with a solid phase extraction manifold and vacuum system. These systems
must be calibrated prior to use, according to vendor or laboratory
specifications. In addition, these liquid handlers have to be used during
laboratory method validation. The liquid handlers that have been shown to
be compatible with this method include the Tomtec® Quadra 3 SPE
(Tomtec, Inc. Hamden, CT), the Caliper Zephyr®, and the Caliper ilOOO
(Caliper Life Sciences, Hopkinton, MA). The selection of these liquid
handlers has typically been based on cost and required sample throughput.
6.5 96-WELL NUNC® DEEP WELL PLATE - 2000 mL plate (Nunc PN# 278752 or
equivalent). Must be compatible with 96-well liquid handler described in Section
6.4.3.
6.6 EXTRACT CONCENTRATION SYSTEM. The 96-well plate requires a
compatible dry-down step for sample pre-concentration following extraction. The
TurboVap™ 96 concentrator evaporator workstation (Zymark® Corp., Hopkinton,
MA) has proven to be well suited for this application, but other evaporator
systems which result in equivalent method performance could be used instead.
Drying should be performed with nitrogen gas with a purity recommended by the
manufacturer.
6.7 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ELECTRON
IONIZATION TANDEM MASS SPECTROMETRY (HPLC/MS/MS) SYSTEM
6.8.1 HPLC COLUMN - Synergi™ 4|im Fusion-RP polar embedded CIS
column (100 x 2.00 mm) (Phenomenex PN OOM-4371-DO or equivalent).
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6.8.2 HPLC SYSTEM - The HPLC system (e.g., Agilent® 1100 HPLC, Agilent
Technologies, Santa Clara, CA; or equivalent) should be equipped with an
autosampler and injector and should provide consistent sample injection
volumes. Mobile phases should be connected to an inline degasser that
runs consistently during sample analysis. The HPLC should be capable of
being configured exactly as described in Table 6-1.
Table 6-1. High Performance Liquid Chromatograph (HPLC) Parameters
Parameter
HPLC Method
Column type
Injection Volume
Autosampler Tray
Temperature
Column Temperature
Injection Settings
Needle Rinse Settings
Typical retention time
Setting
Gradient:
Reservoir A = 10% Methanol in water, 5 mM formic acid
Reservoir B = Acetonitrile, 5 mM formic acid
Time(min) %A %B Flow Rate (jiL/min)
0 93
0.5 93
6.50 70
7.00 70
8.00 70
8.02 40
10.49 40
10.50 93
13.46 93
13.50 93
7 300
7 300
30 300
30 500
30 500
60 500
60 500
7 500
7 500
7 300
Synergi 4(im Fusion-RP polar embedded C18 column (100 x 2.00
mm)
20 nL
4°C
40 ± 5 °C
Draw Speed: 200 uL/min
Eject Speed: 200 uL/min
Injection Mode: Standard
Rinse Solvent: Mobile Phase A
Needle Rinse Time: 10 seconds in
flush port
ABR and ABR internal standard: 2.4 min
RIC and RIC internal standard: 2.8 min
AMAN (native only): 5.1 min
AMAN internal standard: 8.6 min
ABR, abrine; AMAN, alpha-amanitin; HPLC, high performance liquid chromatography; RIC, Ricinine
6.8.3 MASS SPECTROMETER (MS) - The MS/MS (Applied Biosystems API
4000 quadrupole ion trap mass spectrometer, Foster City, CA, or
equivalent) must be capable of performing electron ionization with both
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positive and negative ion detection and has to be configured for multiple
reaction monitoring (MRM) with a dwell time of 100 msec per ion. The
MS/MS was configured as described in Table 6-2.
Table 6-2. Tandem Mass Spectrometer (MS/MS) Parameters
Parameter
MS Scan Mode
lonization Type
Dwell Time
Curtain Gas
Source Temperature
Ion Source Gas 1
Ion Source Gas 2
Collision Gas
Ion Spray Voltage
Entrance Potential
Setting
Multiple Reaction Monitoring (MRM)
Electrospray ionization
100 msec per ion
10
550 (interface heater ON)
75
10
High
5500 in positive polarity mode
-4500 in negative polarity mode
10 in positive polarity mode
-10 in negative polarity mode
The multiple reaction monitoring (MRM) transition conditions for this method
and their associated mass spectrometer settings should be set as stated in Table 6-
3 (ion transitions used in this method are listed in Table 10-1):
Table 6-3. Mass Spectrometer conditions
Analyte
Ricinine Quantitation
Ricinine Confirmation
Ricinine Internal
Standard
Abrine Quantitation
Abrine Confirmation
Abrine Internal Standard
Alpha-Amanitin
Quantitation
Alpha-Amanitin
Confirmation
Alpha-Amanitin Internal
Quantification Standard
Time Post
Injection
(min)
0
0
0
0
0
0
3.5
3.5
3.5
Polarity
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Declustering
Potential
51
51
51
46
31
46
-100
-100
-110
Collision
Energy
41
25
25
17
29
17
-38
-54
-34
Collision
Cell Exit
Potential
8
8
8
12
16
12
-17
-13
-10
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A typical chromatogram is shown in Figure 6-1:
Ricinine
3QQG -
2ODO -
1000 -
4OQG -
2000 -
Q.
u
QJ
+-»
C
Ricinine Internal Standard
Abrine
Abrine Internal Standard
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4
Time (min)
A
a-Amanitin
a-Amanitin Surrogate Internal Standard
4.0
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
Time (min)
Figure 6-1. Chromatogram of typical analyte and internal standard peaks.
7. REAGENTS AND STANDARDS (These reagents were used during the validation of
the method, and only these or their equivalent are acceptable for use. No endorsement
of any supplier or organization should be inferred.)
7.1 GASES, REAGENTS, AND SOLVENTS - Reagent grade or better chemicals
should be used. Unless otherwise indicated, it is intended that all reagents shall
conform to the specifications of the Committee on Analytical Reagents of the
American Chemical Society, where such specifications are available. Other
grades may be used, provided it is first determined that the reagent is of
sufficiently high purity to permit its use without lessening the quality of the
determination.
7.1.1 NITROGEN - 99.9999% pure or better, MS/MS collision cell gas.
10
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7.1.2 REAGENT WATER - purified, deionized water which does not contain
any measurable quantities of the method analyte or interfering compounds
([Tedia®, Fairfield, OH] HPLC or equivalent grade water).
7.1.3 METHANOL - (CH3OH, CAS# 67-56-1) - high purity, demonstrated to
be free of analytes and interferences (Tedia HPLC or equivalent).
7.1.4 ACETONITRILE - (CH3CN, CAS# 75-05-8) - high purity, demonstrated
to be free of analytes and interferences (Tedia HPLC or equivalent).
7.1.5 FORMIC ACID - (HCOOH, CAS# 64-18-6) - reagent grade >95% purity,
demonstrated to be free of analytes and interferences (Sigma®, Sigma-
Aldrich Inc., St. Louis, MO; or equivalent).
7.1.6 SAMPLE PRESERVATION REAGENTS - the following sample
preservation reagents are required for this method and should be
demonstrated to be free of analytes and interferences:
7.1.6.1 SODIUM THIOSULFATE (Na2S2O3, CAS# 7772-98-7) - an
additive used in sample collection (Sigma-Aldrich > 98% pure,
product number 72049 or equivalent).
7.1.6.2 SODIUM OMADINE (C5H4NNaOS, CAS#: 3811-73-2) - an
additive used for sample collection (Sigma-Aldrich > 96% pure,
product number H3261 or equivalent)
7.2 REAGENT PREPARATION
7.2.1 5% METHANOL IN WATER - A 5%/95% (v/v) methanol/water solution
is prepared through volumetric dilution with HPLC grade deionized water.
Measure 10 mL of methanol using an appropriate pipette, volumetric
flask, or graduated cylinder and pour into a clean, dry container with a
capacity of 250 mL or more. Measure 190 mL of HPLC grade deionized
water with a volumetric flask or graduated cylinder and pour into the same
container with the methanol. Mix the solution well.
7.2.2 HPLC MOBILE PHASE A - A 10% methanol solution in HPLC-grade
water with 5 mM formic acid. Measure 100 mL of methanol using an
appropriate pipette, volumetric flask, or graduated cylinder with a 1 L
capacity. Add 700 mL HPLC grade water to the cylinder. Add 192 uL
98% formic acid to the cylinder and fill the cylinder to 1 L with HPLC-
grade water. Pour the solution into a clean dry container with a capacity of
at least 1 L.
7.2.3 HPLC MOBILE PHASE B - A solution of acetonitrile with 5 mM formic
acid. Measure 800 mL of methanol using an appropriate pipette,
11
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volumetric flask, or graduated cylinder with a 1 L capacity. Add 192 uL
98% formic acid to the cylinder and fill the cylinder to 1 L with methanol.
Pour the solution into a clean dry container with a capacity of at least 1 L.
7.3 STANDARDS SOLUTIONS - When a compound purity is assayed to be 96% or
greater, the weight can be used without correction to calculate the concentration
of the stock standard. Solution concentrations listed in this section were used to
develop this method and are included as an example. Standards for sample
fortification generally should be prepared in the smallest volume that can be
measured accurately to minimize the addition of excess organic solvent to
aqueous samples. Store all calibration and control materials at -20 ± 5 °C when
not in use. Even though stability times for standard solutions are suggested in
the following sections, laboratories should use standard QC practices to
determine when their standards need to be replaced.
7.3.1 ISOTOPICALLY LABELLED AND INTERNAL QUANTIFICATION
STANDARD SOLUTIONS - Each analyte requires a separate internal
standard. RIC and ABR internal standards are isotopically-labeled. In this
method, isotopically-labeled internal standards are chemicals that are
structurally identical to the method analyte but substituted with 13C or
13C,D3. These isotopically-labeled internal standards have no potential to
be present in water samples and are not method analytes. The internal
standard for RIC is 13Ce-labeled ricinine and is custom-synthesized by
Cerilliant (Round Rock, TX). The internal stand for ABR is 13C,D3-
labeled L-abrine and it is also custom-synthesized by Cerilliant. The
internal standard for AMAN is structurally similar to AMAN but without
isotopic labeling. The internal standard for AMAN is not known to be
naturally-produced and is not an expected interference for this method.
The internal standard for AMAN is available by custom synthesis through
Invitrogen (Grand Island, NY). These internal standards are added to all
samples, standards, and QC solutions as described in Section 11.1.3.
7.3.2 Prepare or purchase labeled or internal quantification standards for RIC,
ABR, and AMAN. Combine these together to make an internal standard
stock solution.
7.3.2.1 INTERNAL STANDARD STOCK SOLUTION - Combine
internal standards and internal quantification standards in HPLC-
grade water for RIC, ABR, and AMAN so that the final
concentration is approximately 85 ng/mL for RIC and 1265 ng/mL
for ABR IS. The AMAN IQS should be 400 ng/mL. The stock
solution was observed to be stable for at least one year when stored
at -20 ± 5 °C.
7.3.3 ANALYTE STOCK STANDARD SOLUTIONS. Prepare or purchase
individual stock solutions of RIC (Cerilliant, Round Rock, TX or
12
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equivalent), ABR (Cerilliant, Round Rock, TX or equivalent. Please note
that Cerilliant makes this product off-catalogue), and AMAN (Sigma-
Aldrich, St. Louis, MO or equivalent) from a reliable source. These
analytes will be used to generate the stock solutions below. Store these
materials according to manufacturers' instructions.
7.3.3.1 ANALYTE STOCK STANDARD SOLUTION 1 forRIC and
ABR (SS1-RIC/ABR) - Combine RIC and ABR into a single solution
using a 2 mL volumetric flask. To make this solution, weigh out and
reconstitute powdered RIC and ABR in HPLC-grade water or dilute
purchased solutions of RIC and ABR into HPLC-grade water so that the
final concentration of this solution is 10 mg/L RIC and 10 mg/L ABR.
7.3.3.2 ANALYTE STOCK STANDARD SOLUTION 2 for RIC and
ABR (SS2-RIC/ABR) - Dilute SS1-RIC/ABR as follows: combine 100
uL SSI-RIC/ABR with 900 uL HPLC-grade water and mix well. The final
concentration of this solution is 1 mg/L RIC and 1 mg/L ABR.
7.3.3.3 ANALYTE STOCK STANDARD SOLUTION 1 for AMAN
(SSI-AMAN) - Weigh out and reconstitute powdered AMAN in HPLC-
grade water or dilute a purchased solution of AMAN into HPLC-grade
water using a 2 mL volumetric flask so that the final concentration of this
solution is 10 mg/L AMAN.
7.3.3.4 ANALYTE STOCK STANDARD SOLUTION 2 for AMAN
(SS2-AMAN) -Dilute SS1-AMAN as follows: combine 100 uL SS1-
AMAN with 900 uL HPLC-grade water and mix well. The final
concentration of this solution is 1 mg/L AMAN.
7.3.4 CALIBRATION STANDARD STOCK SOLUTIONS - Prepare the
calibration standard stock solutions from dilutions of the analyte stock
solutions in reagent water containing the preservatives specified in section
8.2.1. These standard calibration solutions were observed to be stable for
at least one year when stored at -20 ± 5 °C.
7.3.4.1 PREPARATION OF CALIBRATION STANDARD STOCK
SOLUTIONS - Calibration standard stock solutions may be
prepared using the volumes listed in Table 7-1, below, using a 25
mL volumetric flask. Eight concentrations, along with the numbers
of solutions, are for illustration purposes only. Other
concentrations may be required in practice to meet site-specific
performance and QC goals. (See Section 10.2.3 for considerations
in selecting concentrations of calibration solutions.) All standards
should be diluted with HPLC-grade water.
13
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Table 7-1. Calibration Standard Stock Solution Volumes
Coi
RIC
0
0.5
0.7
1
5
30
50
100
200
icentra
ng/mL
ABR
0
0.5
0.7
1
5
30
50
100
200
tion
)
AMA
0
2
3
5
10
50
100
200
400
Total
Volume
(mL)
25
25
25
25
25
25
25
25
25
Volume of
SS1-
RIC/ABR
75
125
250
500
Analyte St
(V
SS1-
AMAN
125
250
500
1000
ock Standar
L)
SS2-
RIC/ABR
12.5
17.5
25
125
d Solution
SS2-
AMAN
50
75
125
250
ABR, abrine; AMAN, alpha-amanitin; RIC, Ricinine; SS, standard solution
7.3.5 QUALITY CONTROL SOLUTIONS - There are several types of quality
control solutions, some of which are identical in composition but serve
different QC functions and hence may be referred to by different names in
Section 9.
7.3.5.1 SECOND SOURCE QUALITY CONTROL SAMPLE - These
samples are used to verify the accuracy of the calibration standard
solutions (section 7.3.4) and are prepared the same way as the
calibration standards. They are prepared from an analyte source
different from the calibration standard solutions as described more
completely in Section 9.3.5.
7.3.5.2 LABORATORY FORTIFIED BLANKS (LFBs) - LFBs are used
throughout this method for various purposes. The LFB is analyzed
exactly like a sample, and its purpose is to verify that the
methodology is competently replicated, and that the laboratory has
the capability to make accurate and precise measurements. The two
specific LFBs are required in this method are referred to as LFB-
low and LFB-high, which relate to initial and ongoing QC. For the
demonstration of the method in the developer's laboratory, the
LFB-low is 1 ng/mL for RIC, 1 ng/mL for ABR, and 5 ng/mL for
AMAN. The LFB-high for this demonstration is 50 ng/mL for
RIC, 50 ng/mL for ABR, and 100 ng/mL for AMAN. LFB-low and
LFB-high can be prepared as indicated in Table 7-1, in Section
7.3.4. In a particular laboratory, the LFBs should be selected from
similar points in their calibration range (e.g., LFB-low should be
approximately 10 times the MRL (Section 9.2.4) and LFB-high
should be approximately 150 times the MRL).
14
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The LFBs are inherently calibration standards and can be used to
construct the calibration curve. However, the LFBs are specifically
used to develop QC criteria during the initial demonstration of
capability (Section 9.2) and serve as an additional QC function
during each analysis batch. The LFBs serve a similar but generally
more stringent QC function as continuous calibration checks
(Section 10.3).
7.3.5.3 LABORATORY REAGENT BLANK. This blank is prepared as a
LFB with no analyte added (i.e., the 0 ng/mL in Table 7-1).
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 SAMPLE VESSEL PREPARATION FOR COLLECTION
8.1.1 Samples should be collected in a 50-mL polypropylene vessel fitted with a
flat-top polyethylene screw-cap (e.g., BD™ Falcon™ 50 mL centrifuge
tube, BD Inc., Franklin Lakes, NJ; or equivalent).
8.1.2 Vessels should be prepared before sample collection with sodium
thiosulfate and sodium Omadine according to Table 8-1 to fulfill the
purpose(s) listed. The preservatives should be added to all samples,
including initial and on-going QC samples.
8.2 SAMPLE COLLECTION - When sampling from a water tap, samplers should
request guidance about how long to flush the tap, if needed. Depending on site-
specific goals, incident managers may request that the tap not be flushed to
minimize loss of contaminant. If incident managers do not specify a shorter time,
flush until the water temperature has stabilized (approximately 3-5 minutes).
Collect samples from the flowing stream. It may be convenient to collect a bulk
sample in a polypropylene vessel from which to generate individual 50 mL
samples. Keep samples sealed from collection time until analysis. When sampling
from an open body of water, fill the sample container with water from a
representative area. Sampling equipment, including automatic samplers, should be
free of tubing, gaskets, and other parts that may leach interfering analytes into the
water sample. If analyte recovery is unacceptably low, additional experiments
may need to be performed to determine optimal collection conditions.
When filling sample bottles, take care not to flush out the sample preservation
reagents. Samples do not need to be collected headspace free. After collecting the
sample, cap the bottle and agitate by hand to mix the sample with the preservation
reagents. Keep the sample sealed from time of collection until analysis.
8.3 SAMPLE SHIPMENT AND STORAGE - Sample stability was tested at 4 °C. As
a matter of practice, ensure that samples do not experience excessive heat above
15
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this temperature. It is recommended that all samples be iced, frozen (-20 °C), or
refrigerated (4 °C) from the time of collection until extraction. During method
development, no significant differences were observed between standards that
were frozen or refrigerated.
Table 8-1. Preservative Concentrations and Purposes of Preservatives
Mass added to
Compound 50 mL water
sample (mg)
Sodium
thiosulfate
Sodium
Omadine
Concentration in sample „
(g/L) Purp°Se
Dechlorinating
O.Oo
agent
0.064 Microbial inhibitor
8.4 SAMPLE HOLDING TIMES - Table 8-2 presents the RIC, ABR, and AMAN
storage stability data. Samples were collected and stored as described in Section 8
at 4 °C in the presence or absence of preservatives. The data support a 28-day
aqueous holding time for preserved RIC specified in Section 8.4. Preserved ABR
and AMAN and samples can also be held by the laboratory for a maximum of 28
days at 4 °C, although the initial loss of ABR and AMAN (Table 8-2) in some
water occurs within 5 hrs after ABR and AMAN are added to the water sample.
In an actual contamination incident, this 5 hr period will very likely have elapsed
prior to the sample arriving at the laboratory. See section 1.1.1 for discussion of
appropriate application of this method for ABR and AMAN.
8.5 EXTRACT HOLDING TIMES - Water samples should be extracted as soon as
possible but within the holding time (28 days) described in Section 8.4. Data
generated during this study indicate that extracts are stable for at least 28 days
when preserved and stored at 0 °C or lower. In practice, analysis should occur as
soon as possible.
16
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Table 8-2. Recoveries of Ricinine, Abrine, and Alpha-Amanitin in Preservative
Over Time (n=3)
Analyte
1
o
IQ
0)
'3
'0
3
Water Type
Ground Water lb
(chlorine)
Surface Water T
(chlorine)
Surface Water 3d
(monochloramine)
Surface Water 4e
(monochloramine)
Surface Water 5'
(chlorine)
Percent Recovery
15 min
97.1 ±0.8
103.1 ±2.4
94.2 ± 2
110.4 ±9.5
104.3 ±3.4
5hr
105 ±3
103 ±3
102 ±5
113±8
104 ±3
7 days
93 ±5
95 ±3
93 ±11
97 ±6
95 ±4
14 days
99 ±8
102 ±3a
107 ±8
99 ±4
98 ±2
28 days
91 ±10
88 ±1
92 ±7
86 ±10
90 ±6
1
o
in,
at
•c
.a
<
Ground Water lb
(chlorine)
Surface Water T
(chlorine)
Surface Water 3d
(monochloramine)
Surface Water 4e
(monochloramine)
Surface Water 51
(chlorine)
103.6 ±4.4
103.2 ±2.8
103. 8 ±1.5
102.9 ±0.8
102.7 ±1.2
14 ±1
9±0
107 ±3
103 ±3
21 ±1
14 ±0
8±0
98 ±6
100 ±5
21 ±1
14 ±0
9±0a
91±2
97 ±4
20 ±1
14 ±0
8±0
90 ±4
93 ±5
19 ±1
a-Amanitin (100 (J.g/L)
Ground Water lb
(chlorine)
Surface Water T
(chlorine)
Surface Water 3d
(monochloramine)
Surface Water 4e
(monochloramine)
Surface Water 51
(chlorine)
103.6 ±4.4
103.2 ±2.8
103. 8 ±1.5
102.9 ±0.8
102.7 ±1.2
7±1
0±0
97 ±8
104 ±8
30 ±7
6±0
0±0
80 ±5
90 ±5
30 ±10
7±1
0±0a
90 ±20
90 ±20
26 ±6
8±0
0±0
110±30
90 ±10
19 ±1
n (number of replicates) =2 for this data point.
bTotal organic carbon (TOC) below detection limit in well-field; pH 7.6; hardness 500 mg/L; Chlorine 0.2-
0.4 mg/L (monthly averages).
CTOC 0.3 mg/L; pH 8.9; hardness 17 mg/L; Chlorine 1.3 mg/L (monthly average).
dTOC 2.3 mg/L; pH 7.4; hardness 190 mg/L; Monochloramine 3.4 mg/L(monthly averages).
eTOC 7.6 mg/L; pH 9.2; hardness 65 mg/L; Monochloramine 2.4 mg/L (monthly averages).
fTOC 1.0 mg/L; pH 8.5; hardness 130 mg/L; Chlorine 0.8 mg/L (monthly averages).
17
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9. QUALITY CONTROL
9.1 QC requirements include the initial demonstration of capability (IDC) and
ongoing QC requirements that must be met when preparing and analyzing
field samples. This section describes the QC parameters, their required
frequencies, and the performance criteria consistent with typical EPA
quality objectives for drinking water analysis, although these objectives
will be site-specific during a remediation activity. These QC requirements
are considered the minimum acceptable QC criteria in particular for this
method. Laboratories are encouraged to institute additional QC practices
to meet specific needs [18].
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - The IDC must
be successfully performed prior to analyzing any field samples. Prior to
conducting the IDC, the analyst must first generate an acceptable initial
calibration following the procedure outlined in Section 10.2. It should be
noted that the IDC is lengthier than some drinking water methods, but
based on experience in the developer's laboratory, the IDC helps ensure
successful long-term implementation of the method in a variety of other
laboratories. Due to site-specific conditions during an environmental
remediation activity, a shorter IDC may be necessary and appropriate. For
example, a more minimal IDC could consist of: a) demonstration of low
system background (Section 9.2.1); b) 4-7 same-day replicates fortified
near the midrange of the initial calibration curve for precision and
accuracy demonstration, combined with c) the MRL estimation described
in Section 9.2.4. However, QC acceptance requirements, both initial
(Section 9.2.1-9.2.4) and ongoing (Section 9.3), should not be changed. In
addition, a shorter IDC may result in higher QC failure rates and less
accurate quantitation in some concentration ranges. Laboratories should
consider these risks before choosing a shorter IDC.
9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM
BACKGROUND - Any time a new lot of solvents, reagents, and
autosampler vials/plates is used, it must be demonstrated that an
LRB is reasonably free of contamination and that the criteria in
Section 9.3.1 are met.
9.2.2 INITIAL DEMONSTRATION OF PRECISION - Prepare and
analyze at least seven replicates of both laboratory fortified blanks
(LFB-high and LFB-low, see Section 7.3.5.2) over the course of at
least 10 days. Any sample preservative, as described in Section
8.1.2, must be added to these samples. For the initial demonstration
of precision, the relative standard deviation for the concentrations
of the replicate analyses should be less than 20%.
18
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9.2.3 INITIAL DEMONSTRATION OF ACCURACY - Using the
same set of replicate data generated for Section 9.2.2, calculate the
mean recovery. For the initial demonstration of accuracy, the mean
recovery of the replicate values should be within ± 30% of the true
value.
9.2.4 MINIMUM REPORTING LEVEL (MRL) ESTIMATION -
Because cleanup goals will be site-specific, laboratories need to
estimate a minimum reporting level so that incident managers can
understand a specific laboratory's capabilities and can distribute
samples to appropriate laboratories. Establishing the MRL
concentration too low may cause repeated failure of ongoing QC
requirements. If the IDC procedure (Section 9.2.1-9.2.3) is
followed explicitly, establishing the MRL as the lowest standard is
expected to ensure compliance with QC requirements. This
requirement is a result of the rigor of the QC requirements in the
lengthy IDC (Section 9.2.1-9.2.3), especially those associated with
the LFBs (see Section 10.3.3). If a shorter IDC is required by site-
specific conditions (see Section 2.2), the MRL should be
confirmed with the procedure below.
9.2.4. 1 Fortify and analyze seven replicate LFBs at the proposed
MRL concentration. These LFBs must contain all method
preservatives described in Section 8.1.2. Calculate the
mean measured concentration (Mean) and standard
deviation for the method analytes in these replicates.
Determine the half range for the prediction interval of
results (HRpiR) for each analyte using the equation below:
HRpm = 3.963s
where s is the standard deviation and 3.963 is a constant
value for seven replicates.
9.2.4.2 Confirm that the upper and lower limits for the prediction
interval of the result (PIR = Mean +_ HRpm) meet the upper
and lower recovery limits as shown below:
The Upper Predicted Interval of Results (PIR) Limit should be
<150% recovery.
xlo0o/o<150o/o
FortifiedConcentration
19
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The Lower PIR Limit should be > 50% recovery.
Mean-HRPIR
FortifiedConcentration
9.2.4.3 The MRL is validated if both the upper and lower PIR
limits meet the criteria described above (Section 9.2.4.2). If
these criteria are not met, the MRL has been set too low
and should be confirmed again at a higher concentration.
9.2.5 CALIBRATION CONFIRMATION - The calibration is
confirmed by analysis of a second source quality control sample as
described in Section 9.3.5
9.2.6 DETECTION LIMIT (DL). The detection limit is a statistical
determination of precision, and accurate quantitation is not
expected at the DL. Replicate analyses for this procedure should be
done over at least three days (i.e., both the sample preparation and
the HPLC/MS/MS analyses should be done over at least
three days). At least seven replicate LFBs should be analyzed
during this time period. The concentration may be estimated by
selecting a concentration that is between 3 and 5 times the signal-
to-noise ratio for the analyte peak. The appropriate fortification
concentrations will be dependent upon the sensitivity of the
HPLC/MS/MS system used. Any preservation reagents added in
Section 8.1.2 must also be added to these samples. Note that the
concentration for some IDC steps may be appropriate for DL
determination, in which case the IDC data may be used to calculate
the DL. (For example, for the results presented in Section 13, eight
replicate LFBs were analyzed over 10 days with two LFBs
individually fortified on day one, two LFBs individually fortified
on day three, and two LFBs individually fortified on day five, etc.).
Analyze the replicates through all steps of Section 11. Calculate
the DL from the equation: DL = s x t(n.\)
where:
s = standard deviation of replicate analysis, without subtraction of values
of analyte-free blanks
t = Student's t value for the 99% confidence level with n-1 degrees of
freedom
n = number of replicates.
20
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9.3 ONGOING QC REQUIREMENTS - This section summarizes the
ongoing QC criteria when processing and analyzing field samples. The
required QC samples for an analysis batch include the laboratory reagent
blank (LRB) and four continuing calibration check (CCC) solutions.
9.3.1 LABORATORY REAGENT BLANK (LRB) - An LRB is
required with each analysis batch (Section 3.1) to confirm that
potential background contaminants are not interfering with the
identification or quantitation of method analytes. Running the LRB
first may prevent unnecessary analysis if the LRB is invalid.
Preparation of the LRB is described in Section 7.3.5.2. If the LRB
produces a peak within the retention time window of the analyte,
accurate determination of the analyte will not be possible.
Determine the source of contamination and eliminate the
interference before processing samples. Background contamination
must be reduced to an acceptable level before proceeding.
Background from method analytes or other contaminants that
interfere with the measurement of the method analyte should be
below 1/3 of the MRL. Blank contamination is estimated by
extrapolation, if the concentration is below the lowest calibration
standard. This extrapolation procedure is not allowed for sample
results as it may not meet data quality objectives. If the method
analytes are detected in the LRB at concentrations greater than 1/3
the MRL, then all data for the problem analyte(s) are considered
invalid for all samples in the analysis batch.
9.3.2 ONGOING CALIBRATION. During development of this method,
the analytical system was recalibrated at the beginning of each
analysis batch using the same analyte concentrations determined
during the initial calibration. The acceptance criteria for the
ongoing calibration are described in Section 10.2.5, except that
removal of calibration points may result in too few calibration
points and therefore an invalid calibration. The ongoing calibration
is performed after the first two continuing calibration check (CCC)
samples (Sec. 9.3.3) to allow for corrective action if the calibration
fails.
As mentioned in Section 2.2, in some well-considered
circumstances and in consultation with the sample submitter about
increased QC and quantitation risk, it may be desirable not to
perform recalibration at the beginning of each analysis batch and
instead rely on CCC samples (as described in Section 9.3.3) to
verify ongoing calibration. If so, the beginning CCC of each
analysis batch should be at or below the MRL to verify instrument
sensitivity prior to any analyses. Subsequent CCCs should
21
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alternate between a medium and high concentration calibration
standard.
9.3.3 CONTINUING CALIBRATION CHECK (CCC) - CCC standards
containing the preservatives, if any, are analyzed at the beginning
of each analysis batch and after every 20 field samples. Note that
there are up to four CCCs depending on the IDC appropriate for
the site specific circumstance. In the lengthier IDC described in
Section 9.2, there are four CCCs: LFB-low and LFB-high, which
are analyzed before the batch, and the lowest and highest
calibration standards from the ongoing calibration, which are
analyzed after the field samples. If a different IDC approach was
used based on site-specific goals and tolerance of QC and
quantitation risk, it may acceptable to run only one of these
calibration standards as the CCC before and after the batch. If so,
the beginning CCC of each analysis batch should be at or below
the MRL in order to verify instrument sensitivity prior to any
analyses. Subsequent CCCs should alternate between a medium
and high concentration calibration standard. See Section 10.3 for
acceptance criteria for the various CCCs. Preparation of the CCCs
is described in Section 7.3.5.
9.3.4 LABORATORY FORTIFIED BLANK (LFB) - Since this method
utilizes procedural calibration standards, which are fortified
reagent waters, there is no difference between the LFB and the
CCC, except for the order in which they are run as part of an
analysis batch and the corresponding QC acceptance criteria. The
acronym LFB is used for clarity in the IDC.
9.3.5 SECOND SOURCE QUALITY CONTROL SAMPLES (QCS) -
As part of the IDC (Section 9.2), each time a new analyte stock
standard solution 1 for RIC/ABR or AMAN (SS1-RIC/ABR or
SS1-AMAN, Section 7.3.3.1) is prepared, and, at least quarterly,
analyze a QCS from a source different from the source of the
calibration standards. If a second vendor is not available, then a
different lot of the standard should be used. The QCS should be
prepared near the midpoint of the calibration range and analyzed as
a CCC. Acceptance criteria for the QCS are identical to the CCCs;
the calculated amount for each analyte should be ± 30% of the
expected value. If measured analyte concentrations are not of
acceptable accuracy, check the entire analytical procedure to locate
and correct the problem.
9.3.6 INTERNAL STANDARD (IS) or INTERNAL
QUANTIFICATION STANDARD (IQS) - The analyst should
monitor the peak area of the IS or IQS in all injections during each
22
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analysis day. The IS or IQS peak area must meet the criteria in
both of the following two subsections.
9.3.6.1 The internal standard should produce a peak area at least
five times higher than the peak area of the quantitation ion
transition of the corresponding analyte in the lowest
concentration calibration solution. If it does not, the
concentration of IS or IQS may not be as predicted. Prepare
new calibration solutions, QC samples, and field samples
with an appropriately increased concentration of IS or IQS.
9.3.6.2 The IS or IQS response (peak area) in any sample must not
deviate from the response in the most recent CCC by more
than 30%, and must not deviate by more than 50% from the
area measured during initial analyte calibration. If the IS or
IQS area in a chromatographic run does not meet these
criteria, inject a second aliquot of that extract.
9.3.6.2.1 If the reinjected aliquot produces an acceptable
IS or IQS response, report results for that aliquot.
9.3.6.2.2 If the reinjected aliquot fails the IS or IQS
criterion, the analyst should check the calibration by
reanalyzing the most recently acceptable calibration
standard. If the calibration standard fails the criteria
of Section 10.3, recalibration is in order per Section
10.2. If the calibration standard is acceptable, report
results obtained from the reinjected aliquot, but
annotate as "suspect/IS(IQS) recovery."
Alternatively, prepare another aliquot of the sample
as specified in Section 11.2 or collect a new sample
and re-analyze.
9.3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) and
LFSM DUPLICATES (LFSMD) - The isotopically-labeled and
internal quantification standards in this method also serve the role
of the LFSM, which is used to determine that the sample matrix
does not adversely affect method accuracy. In the context of
application of this method for environmental remediation, it is not
expected that there would be native RIC, ABR, or AMAN
background concentrations. Also, it is likely that the water samples
will come from the same drinking water system, and hence the
sample matrices from a single collection time will be very similar.
Further, experience with the automated extraction equipment used
suggests that most failures in IS QC requirements result from
23
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failure of the automation equipment. This situation would apply to
LFSM failure, as well. Accordingly, neither LFSMs nor duplicate
LFSMs would be expected to yield additional information about
influence of sample matrix on method accuracy, except for the
unlikely case of a feature of the sampling/remediation plan that
produces a co-eluting peak with chromatographic and mass
spectral properties identical to RIC, ABR, or AMAN. In this case,
the laboratory should discuss with the submitter the number and
frequency of LFSMs.
9.3.7.1 If an LFSM and LFSMD are deemed necessary, calculate
the relative percent difference (RPD) for duplicate LFSMs
(LFSM and LFSMD) using the equation
\LFSM-LFSMD\
= ^ ^
(LFSM+LFSMD} 12
9.3.7.2 Relative percent difference (RPD) for duplicate LFSMs
should be <30% for samples fortified at or above their
native concentration. Greater variability may be observed
when LFSMs are fortified at analyte concentrations that are
within a factor of two of the MRL. LFSMs fortified at these
concentrations should have RPDs that are <50%. If the
RPD of any analyte falls outside the designated range, and
the laboratory performance for that analyte is shown to be
in control in the CCC, the recovery is judged to be matrix
biased. The result for that analyte in the unfortified sample
is labeled "suspect/matrix" to inform the data user that the
results are suspect due to matrix effects.
9.3.8 FIELD DUPLICATE (FD) - Field duplicates check the precision
associated with sample collection, preservation, storage, and
laboratory procedures. Some of these factors are out of control of
the laboratory, and the rest are covered by other QC checks.
Accordingly, results of any field duplicates requested should be
discussed with the sample submitter if they do not meet the
following criteria:
9.3.8.1 Calculate the relative percent difference (RPD) for
duplicate samples (FD1 and FD2) using the equation
24
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FDl-FD2
(FDl + FD2)/2
lOO
9.3.8.2 RPDs for FDs should be <30%. Greater variability may be
observed when FDs have analyte concentrations that are
within a factor of two of the MRL. At these concentrations,
FDs should have RPDs that are <50%. If the RPD of any
analyte falls outside of the designated range, and the
laboratory performance for that analyte is shown to be in
control in the CCC, the recovery is judged to be biased.
The result for that analyte in the unfortified sample is
labeled "suspect/field duplicate bias" to inform the data
user that the results are suspect due to field bias. (Note
some other sources of laboratory bias may also be present.)
10. CALIBRATION AND STANDARDIZATION
10.1 All laboratory equipment should be calibrated according to manufacturers'
protocols and equipment with expired calibrations should not be used.
Demonstration and documentation of acceptable mass spectrometer tune
and initial calibration is required before any samples are analyzed. After
the initial calibration is successful, the instrument is recalibrated using the
same conditions as the initial calibration before each analysis batch.
(Ongoing calibration is discussed in section 9.3.3.) Verification of mass
spectrometer tune should be repeated each time a major instrument
modification is made or maintenance is performed and prior to analyte
calibration.
10.2 INITIAL CALIBRATION
10.2.1 ELECTROSPRAY IONIZATION (ESI)-MS/MS TUNE
10.2.1.1 Calibrate the mass scale of the MS with the
calibration compounds and procedures prescribed by the
manufacturer.
10.2.1.2 Optimize the [M+HJ+ for each method analyte and
surrogate or labeled internal standard by infusing
approximately 1.0-5.0 ug/mL of each analyte (prepared in
water containing 15% methanol) directly into the MS at the
chosen LC mobile phase flow rate (between 0.3 and 0.5
mL/min). Each analyte should be tuned separately using
solutions containing only one analyte, internal
quantification standard, or labeled internal standard. The
MS parameters (voltages, temperatures, gas flows, etc.) are
25
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varied until optimal analyte responses are determined. The
method analytes may have different optima requiring some
compromise between the optima. See Tables 6-2 and 6-3
for ESI-MS/MS conditions used in method development
10.2.1.3 Optimize product ions for each analyte, IS, or IQS
by infusing approximately 1.0-5.0 ug/mL of each analyte
(prepared in the initial mobile phase conditions) directly
into the MS at the chosen LC mobile phase flow rate
(between 0.3 and 0.5 mL/min). This tune can be done on a
mix of the method analytes. The MS/MS parameters
(collision gas pressure, collision energy, etc.) are varied
until optimal analyte responses are determined. The
MS/MS product ions used in this method are listed in Table
10-1. See Tables 6-2 and 6-3 for MS/MS conditions used in
method development.
Table 10-1. Ion transitions monitored for Ricinine, Abrine, and Alpha-Amanitin
Analyte
Ricinine Analyte
Internal Standard
Abrine Analyte
Internal Standard
Alpha-Amanitin Analyte
Internal Quantification
Standard
Precursor Ion
(m/z)
165
171
219
223
917.4
887.5
Quantitation Ion
(m/z)
82
144
188
188
899.4
843.5
Confirmation Ion
(m/z)
138
-
132
-
560.2
-
10.2.1.4 Establish LC operating parameters that optimize
resolution and peak shape. LC conditions used in method
development can be found in Table 6-1. The LC conditions
listed in Table 6-1 may not be optimum for all LC systems
and may need to be optimized by the analyst. If possible,
optimize chromatographic conditions so that a unique
quantitation ion is available for each analyte that is free
from interference due to an identical product ion in any co-
eluting (or overlapping) peak(s).
10.2.2 INSTRUMENT CONDITIONS - Operational conditions are
tabulated in Section 6.8.3. Alteration of the conditions is not
recommended and would require redevelopment of QC criteria.
Frequently reported problems can be avoided by: (1) checking that
needle wash solutions are adequately filled and the injection
26
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syringe is functioning properly and (2) changing the septum as
needed.
10.2.3 Prepare six calibration standards as described in Section 7.3.4.
Note that, as procedural calibration standards, they are processed
through the procedure in Section 11, in which isotopically-labeled
internal standard or internal quantification standard is added before
extraction. In practice, the lowest concentration of the calibration
standard should be at or below the MRL (Section 9.2.4), which
will depend on system sensitivity. The lowest point on the
calibration curve is close to the reported detection limit and the
highest point is above the expected range of results. The remaining
points are distributed between these two extremes, with the
majority of points in the concentration range where most
unknowns are expected to fall.
10.2.4 The HPLC/MS/MS system is calibrated using the internal standard
technique, as implemented by the data system software. Construct
a calibration curve using at least a six-point curve of response
ratios (i.e., ratio of calibration standard peak area to internal
standard peak area). As the internal standard concentration is
consistent among samples and calibrators, some laboratories,
including the developers of this method, have found it convenient
to set it to a value of one instead of the actual concentration.
10.2.5 CALIBRATION ACCEPTANCE -- Calculate the slope and
intercept of the calibration curve with 1/x weighting (or other
appropriate weighting) by a linear least squares fit (or other
appropriate calibration function). Evaluate the r2 value for the
curve, which must be greater than 0.980. Linearity of the standard
curve should extend over the entire standard range. Each calibration
point, except the lowest point, for the analyte should calculate to be
70 to 130 percent of its true value. The lowest point should calculate
to be 50 to 150 percent of its true value. If these criteria cannot be
met, the analyst will have difficulty meeting ongoing QC criteria.
If any standard is in error and does not fit the standard curve (i.e.,
the r2 value for the curve is < 0.980), this standard can be removed
from the calibration. No more than one standard may be discarded
in any given calibration curve. If either the high or low standard is
dropped, the reporting limits must be adjusted accordingly. The
resulting r2 value should be greater than 0.980.
10.3 CONTINUING CALIBRATION CHECKS (CCCs). As described in
Sections 9.3.2 and 9.3.3, up to four CCCs are used in conjunction with
each analysis batch, depending on initial IDC approach and site specific
data quality objectives. The LRBs, LFBs, and CCCs are not counted as the
20 samples that constitute an analysis batch.
27
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10.3.1 Inject an aliquot of the appropriate concentration calibration
solution and analyze with the same conditions used during the
initial calibration.
10.3.2 Acceptance of the calibration solutions is based on the same
criteria as described in Section 10.2.5. Failure to meet these criteria
is a rare occurrence and suggests that maintenance of the
HPLC/MS/MS system is required.
10.3.3 Acceptance of the results of the LFB-low and LFB-high is based
on the quality control limits (Section 10.3.3.1) established via the
IDC. Acceptability of results for that entire analytical batch is
dependent upon the agreement of the results from these control
materials within established ranges. Quality control limits for the
CCCs are based primarily on the standard deviation (on-i, sigma)
of the replicate analysis in the IDC (Section 9.2.2). Section 13.3
presents sample values for these parameters obtained in the
developer's laboratory, in which eight replicate analyses performed
over no less than 10 days are used to establish the LFB-low and
LFB-high limits (Section 9.2.2). If the CCC results do not meet the
following criteria, the CCC is "out-of-control," and the cause of
the failure should be determined and corrected. No results from the
associated analytical batch may be reported. These criteria apply to
non-zero analyte concentrations used to make the quality control
LFB solutions in section 7.3.5.2.
10.3.3.1 If both the LFB-low and LFB-high results are within
two times the standard deviation of the mean
determined during the IDC, then accept the entire
analytical batch. Otherwise, reject the entire analytical
batch.
10.3.4 Common remedial actions if the CCCs fails to meet acceptable
criteria
10.3.4.1 LOW ANALYTE RESPONSE - If the signal-to-noise
ration of the low standard confirmation ion falls below
10, this signal level indicates that the instrumental
sensitivity or SPE recovery has fallen below acceptable
limits. The following steps should be taken and the
instrument sensitivity rechecked after each corrective
action is performed. Once sensitivity has been
reestablished, further steps are not necessary.
28
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i. Re-extract the samples.
ii. If peak tailing or fronting is a significant issue, replace the
HPLC column.
iii. Ensure the source of the MS/MS is clean.
iv. Clean the mass spectrometer source plate.
v. Flush all tubing on the HPLC/MS/MS instrument with
95%/5% acetonitrile/water for 15 minutes followed by 5
minutes of equilibration with 5%/95% acetonitrile/water.
10.3.4.2 Analyte in standards - If an inordinately large amount
of analyte is measured in one of the calibration
standards, but this large amount of analyte is not seen in
the remainder of the calibration samples, contamination
of this particular sample is indicated. The source of this
incident should be investigated to prevent repeat
occurrences, but no further action is required. The
contaminated calibration standard should be excluded
when developing the calibration curve.
10.3.4.3 Analyte in all samples - If an inordinately large amount
of analyte is present in all measurements for a particular
day, it is likely that one or more of the spiking solutions
is contaminated. If necessary, prepare new solutions.
11. PROCEDURE
11.1 SAMPLE PREPARATION
11.1.1 Samples are collected, preserved and stored as presented in Section
8. Allow samples to come to room temperature prior to analysis.
11.1.2 If using a TurboVap 96 evaporator system, set it to 50-60 °C.
Follow manufacturers' directions for other equipment.
Note: Steps 11.1.3 through 11.1.8 can be performed using an automated
liquid handler or a manual pipettor with a manual 96-well manifold.
However, data presented in this document were collected using an
automated liquid handler.
11.1.3 Fill 96 plate wells.
29
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11.1.3.1 Into each well of the 96-well Nunc deep well plate
(Nalge Nunc International, Rochester, NY), add 30 uL
of the RIC/ABR/AMAN internal standard mixture (refer
to section 7.3.2).
11.1.3.2 Into each sample well, add 200 uL of sample.
11.1.3.3 Into each blank well, add 200 uL of reagent water (for
the LRB).
11.1.3.4 Into each calibration standard well, add 200 uL of
RIC/ABR/AMAN calibration standard stock solutions
(refer to sections 7.3.4).
11.1.3.5 Into each quality control well, add 200 uL of appropriate
quality control material (refer to section 7.3.5).
11.1.4 Mix on the plate shaker for 2 min or mix by other appropriate
means.
11.1.5 Plate SPE procedure: For each well on the Nunc plate filled in
Section 11.1.3, perform the following steps and do not let wells go
dry for more than 1 minute:
11.1.5.1 Condition the selected well on the Phenomenex Strata-X
60-mg SPE well plate (Phenomenex, Torrance, CA) with
1125 uL of 100% methanol.
11.1.5.2 Condition the SPE plate with 1125 uL of deionized
water.
11.1.5.3 Load 150 uL from the Nunc sample plate and draw
through the SPE plate using positive or negative pressure.
11.1.5.4 Wash the SPE plate with 1125 uL of 5% methanol/95%
water (Section 7.2.1).
11.1.5.5 Elute the sample with 800 uL of acetonitrile into a 96-
well Nunc deep well plate.
11.1.6 Blow the sample to dryness using a gentle stream of nitrogen gas
at 50-60 °C. If using a TurboVap evaporator system, set the flow
rate to 40 flow units until approximately 50% has been evaporated.
Then raise the flow rate to 50-75 flow units until dry. When using
30
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systems other than the TurboVap, set the flow rate for the blow
down gas according to manufacturer's directions.
11.1.7 Add 100 uL of HPLC-grade water to reconstitute each sample and
vortex.
11.1.8 Transfer the water solution into appropriate autosampler vials or
a 96-well autosampler plate.
11.2 ANALYSIS OF SAMPLE EXTRACTS
11.2.1 Establish operating conditions as described in Section 10.2.2.
11.2.2 Establish a valid initial calibration following the procedures
outlined in Section 10.2 or confirm that the calibration is still valid
by running both CCCs as described in Section 10.3. If establishing
an initial calibration for the first time, complete the IDC as
described in Section 9.2.
11.2.3 Set up the available automation equipment and software as
specified by the manufacturer for batch analysis, paying particular
attention to the following potential sources of errors/complications:
11.2.3.1 On the instrument computer, edit the automation
software:
(a) Select the sample type.
(b) Identify the correct vial position.
(c) Name the sample. Due to large number of samples
analyzable with the automation equipment, it is important
that appropriate record keeping (e.g., database, notebooks,
data files) should be used to track specimens.
(d) Enter information related to particular specimens into
the software manually or by electronic transfer.
(e) Select the instrument control method.
(f) Identify the target path where the data will be stored.
11.2.3.2 Check to be sure that the number and positions of
samples entered on the sequence setup page correspond
to the samples in the autosampler.
11.2.4 Run the automation sequence to analyze the batch of aliquots of
field and QC samples at appropriate frequencies (Section 9, 10.3).
All field, QC, and calibration standards should be run using the
same HPLC/MS/MS conditions. At the conclusion of data
acquisition, use the same software that was used in the calibration
procedure to identify the peaks in predetermined retention time
31
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windows of interest. Use the data system software to examine the
ion abundances of components of the chromatogram.
11.2.5 COMPOUND IDENTIFICATION - The presumed peaks for RIC,
ABR, and AMAN in the sample must appear in the same retention
time window as the internal standard corresponding to each analyte
(around 2.8 min for RIC, 2.4 min for ABR, and 5.1 min for AMAN
in the developer's laboratory) and have similar chromatographic
characteristics such as peak shape. This compound identification
relies on expert judgment of the analyst because the retention times
reported by the software are not always reliable. Identification of
the peak as RIC, ABR, or AMAN is then confirmed through
calculating the confirmation ratio (CR), i.e., by dividing the
response for confirmation transition by the response for
quantitation transition of the presumed analyte peak. Using the
manufacturer's software or manually, compare the confirmation
ratio of the peak from the sample with the mean of the CRs
measured for the six calibration standards associated with that
batch. The mean CR is the average CR from the calibration
standards only and is batch dependent. The CR value for each
sample should be within 30% of the mean. (CR value was
approximately 3.02 for RIC, 0.43 for ABR, and 0.13 for AMAN in
the developer's laboratory.) This percent tolerance is based on
running a new calibration curve with each batch. Depending on
site-specific data quality objectives, this might not have been done.
In this case, the CR should still be within 30% of the most recent
calibration, which should have been analyzed no more than 30
days prior. Furthermore, no components of the analytical
instrument should have been changed or recalibrated between the
most recent CCC and analysis of the current batch of samples. Any
changes to the instrument could result in this tolerance being
exceeded.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Concentrations are calculated using the ions listed in Table 10-1. Use of
other ions is not advised. If a particular instrument cannot produce the
fragments listed in Table 10-1, that instrument should not be used to run
this method.
12.2 Calculate analyte concentrations using the ongoing multipoint calibration
established in Section 9.3.2. Do not perform calibration using just the
CCC or LFB-low and LFB-high data to quantitate analytes in samples,
although these samples might be part of the ongoing calibration curve.
32
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12.3 All raw data files are quantified using the quantitation capabilities of the
instrument software. The peaks are automatically integrated using the
software-associated integration program, and the integration of each peak
is reviewed and manually corrected as appropriate and consistent with
laboratory policy. This process is particularly important for the calibration
standards. The quality control samples (e.g., CCCs and LFBs) are
quantified and evaluated against the calibration curve, and each field
sample is then quantified against that calibration curve. The run data can
be processed within instrument data analysis software and exported to
external spreadsheets, per laboratory policy, generating files containing
the unknown and QC concentrations, retention times, standard curves, and
other run information.
12.3.1 Results are generally reported to two significant digits. In addition
to analytical measurements of unknowns, statistical results of
measurement of blanks should accompany all results.
12.3.2 Check all sample and analytical data for transcription errors and
overall validity after being entered into the instrument software
database. Back up both the instrument and data storage databases
onto external media according to individual laboratory guidelines.
13. METHOD PERFORMANCE
13.1 ANALYTICAL IDENTIFICATION-Analyte identification using the
approach described in Section 11.2.5 resulted in no false positives or
negatives for the samples reported below. There was very low background
noise according to the signal-to-noise ratios for the ion transitions
monitored.
13.2 SINGLE LABORATY MINIMUM REPORTING LEVELS and
DETECTION LIMIT- The reportable range of results for RIC, ABR, and
AMAN are summarized in Table 13-1, along with the DL determined
from the IDC procedure described previously. The lowest calibration
standard is used as the method reportable limit (MRL) and the DL
calculated from the standard deviation of replicate measurements of that
standard. The highest reportable limit (HRL) is based on the highest linear
calibration standard.
33
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Table 13-1. Method Performance (n=8 over 10 days)
Analyte
Ricinine
Abrine
Alpha-Amanitin
DL
(HS/L)
0.09
0.06
0.54
MRL
(HS/L)
0.50
0.50
2.0
HRL
(HS/L)
200
200
400
DL, detection limit; HRL, highest reportable limit; MRL, minimum reportable level; n,
number of replicates
13.3 SINGLE LABORATORY ACCURACY AND PRECISION for LFBs -
Single laboratory precision and accuracy data are represented in Table 13-
2. Accuracy is defined as the mean of the measured concentration in the
fortified samples divided by the fortification concentration, expressed as a
percentage. Method accuracy was determined by analyzing LFBs at the
two non-zero levels in Section 7.3 (i.e., LBF-low and -high) and at least
seven replicates for each of the two concentration levels over a period of
10 days for each analyte. The means, standard deviations, and relative
standard deviations for the two LFBs are shown in Table 13-2.
Table 13-2. Single Lab Precision and Accuracy (n=8 over 10 days).
Analyte
Ricinine
Abrine
Alpha-Amanitin
QC Pool
LFB-Low
LFB-High
LFB-Low
LFB-High
LFB-Low
LFB-High
Fortified
Cone.
(re/L)
1.0
50
1.0
50
5.0
100
Mean of
IDC
Replicates
(|ig/L)
0.93
50
0.97
51
5.0
101
Standard
Deviation
(Mg/L)
0.05
0.68
0.04
0.45
0.40
10.2
RSD
(%)
5.4
1.4
4.1
0.88
8.0
10.1
Measured
Cone.
(%)a
93 ± 5
100 ± 1
97 ±4
102 ± 1
100 ±8
101 ± 10
The measured concentration is expressed as a percent of that fortified (x ± on-i).
IDC, initial demonstration of capability; LFB, laboratory fortified blank; n, number of replicates; QC,
quality control; RSD, relative standard deviation
13.4 SINGLE LABORATORY RECOVERY AND PRECISION FOR TAP
WATER MATRICES. Table 8-2 expresses percent mean recoveries for
RIC, ABR, and AMAN in several different chlorinated and chloraminated
tap waters derived from the types of sources (i.e., ground or surface water)
indicated. Water quality parameters describing these sources are indicated
in the footnotes. Percent recoveries were determined by dividing the
measured concentration by the spiked concentration (number of replicates
34
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(n)=3). No analytes or interferences were detected in the unspiked
samples.
13.5 SPECIAL NOTE FOR ABR AND AMAN. The performance data for the
method presented in Section 13 are from samples preserved as described
in Section 8.1.2. As indicated in Section 8.4, ABR and AMAN exhibit an
initial analytical signal loss that is dependent on the specific drinking
water. Accordingly, if ABR and AMAN are unexpectedly low (or absent)
for a particular drinking water, additional QC steps, such as additional
LFSMs held at least 5 hours (see Section. 9.3.7), maybe required.
14. POLLUTION PREVENTION
14.1 This method utilizes solid phase extraction to extract analytes from water.
The method requires the use of reduced volumes of organic solvent and
very small quantities of pure analytes, thereby minimizing the potential
hazards to both the analyst and the environment as compared to the use of
large volumes of organic solvents in conventional liquid-liquid
extractions.
14.2 For information about pollution prevention that may be applicable to
laboratory operations, consult "Less is Better: Laboratory Chemical
Management for Waste Reduction" available from the American Chemical
Society's Department of Government Relations and Science Policy on-line
at
http://portal.acs.org/portal/fileFetch/CAVPCP_012290/pdfAVPCP_012290
.pdf (accessed May 2010).
15. WASTE MANAGEMENT
15.1 Dispose of waste materials in compliance with the individual laboratory's
chemical hygiene plan, as well as federal, state, and local regulations.
Always dispose of solvents and reagents in an appropriate container
clearly marked for waste products and store them temporarily in a
chemical fume hood. Dispose of RIC, ABR, and AMAN in an appropriate
waste stream as well.
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16. REFERENCES
[1] "Drinking Water Contaminants: National Primary Drinking Water Regulations,"
US Environmental Protection Agency, 2012.
http://water.epa.gov/drink/contaminants/index.cfm, accessed July 2012.
[2] J.L. Knaack, C.T. Pittman, J.V. Wooten, J.T. Jacob, M. Magnuson, E. Silvestri,
R.C. Johnson, "Quantitative analysis of ricinine, abrine, and alpha-Amanitin in
finished tap water." (manuscript under preparation).
[3] CDC, NIOSH emergency response card: ricin.
http://www.cdc.gov/NIOSH/ershdb/EmergencyResponseCard_29750002.html,
accessed June 2012.
[4] S. Olsnes, J.V. Kozlov, "Ricin." Toxicon 39 (2001) 1723-1728.
[5] CDC, "NIOSH Emergency Response Card: Abrin." NIOSH Emergency Response
Card: Abrin,
http://www.cdc.gov/NIOSH/ershdb/EmergencvResponseCard 29750000.html.
accessed July 2012 (2008).
[6] S. Olsnes, "The history of ricin, abrin and related toxins." Toxicon 44.4 (2004)
361-370.
[7] R.C. Johnson, S.W. Lemire, A.R. Woolfitt, M. Ospina, K.P. Preston, C.T. Olson,
J.R. Barr, "Quantification of ricinine in rat and human urine: A biomarker for
ricin exposure." J Anal Toxicol 29 (2005) 149-155.
[8] R.C. Johnson, Y.T. Zhou, R. Jain, S.W. Lemire, S. Fox, P. Sabourin, J.R. Barr,
"Quantification of L-abrine in human and rat urine: a biomarker for the toxin
abrin." J Anal Toxicol 33.2 (2009) 77-84.
[9] J. Owens, C. Koester, "Quantitation of abrine, an indole alkaloid marker of the
toxic glycoproteins abrin, by liquid chromatography/tandem mass spectrometry
when spiked into various beverages." J Agr Food Chem 56.23 (2008) 11139-
11143.
[10] J. Rittgen, M. Putz, U. Pyell, "Identification of toxic oligopeptides in Amanita
fungi employing capillary electrophoresis-electrospray ionization-mass
spectrometry with positive and negative ion detection." Electrophoresis 29.10
(2008)2094-2100.
[11] J. Vetter, "Toxins of Amanita phalloides" Toxicon 36.1 (1998) 13-24.
[12] "Carcinogens - working with carcinogens," Department of Health, Education,
and Welfare, Public Health Service, Center for Disease Control, National Institute
for Occupational Safety and Health, Publication No. 77-206, August 1977.
[13] "OSHA Safety and Health Standards, General Industry," (29CFR1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised, January
1976).
[14] "Safety in academic chemistry laboratories," 7th Edition, Volume 2. (2003)
American Chemical Society Publication, Committee on Chemical Safety:
Washington, D.C.
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[15] S.C. McGrath, D.M. Schieltz, L.G. McWilliams, J.L. Pirkle, J.R. Barr, "Detection
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