oo a
VOLATILE ORGANIC COMPOUNDS IN WHOLE BLOOD - DETERMINATION BY
HEATED DYNAMIC HEADSPACE PURGE AND TRAP
ISOTOPE DILUTION GC/MS
•by
Paul H. Cramer
Kathy E. Boggess
John M. Hosenfeld
EPA Contract No. 68-02-4252
MRI Project No. 8822-A(01)
July 24, 1987
For
National Human Monitoring Program
U.S. Environmental Protection Agency
Field Studies Branch
Design and Development Branch
Office of Pesticides and Toxic Substances
401 M Street, S.W.
Washington, DC 20460
Attn: Ms. Janet Remmers and Mr. Philip Robinson, Work Assignment Managers
Dr. Joseph Breen and Ms. Cindy Stroup, Program Managers
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DISCLAIMER
This document has been reviewed and approved for publication by the Office of
Toxic Substances, Office of Pesticides and Toxic Substances, U.S. Environmental
Protection Agency. The use of trade names of commercial products does not
constitute Agency endorsement or recommendation for use.
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PREFACE
This method was developed under EPA Contract Nos. 68-02-3938 and
68-02-4252 for the Field Studies Branch in the Office of Toxic Substances.
The method is primarily intended to be used in the National Blood Network
to establish the baseline levels of volatile organics in the general popu-
lation of the United States.
This method was developed by Mr. Paul Cramer and Ms. Kathy Boggess.
Mr. John M. Hosenfeld was the MRI Work Assignment Leader. We gratefully
acknowledge the valuable assistance given by Ms. Janet Remmers, EPA Work
Assignment Manager and Dr. Joseph Breen, EPA Project Officer.
MIDWEST RESEARCH INSTITUTE
Paul H. Cramer
Assistant Work Assignment Leader
]ohn M. Hosenfeld
Work Assignment Leader
Approved:
Jack Balsinger
Quality Assurance Coordinator
Paul C. Constant
Program Manager
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1.0 Scope and Application
1.1 This method covers the determination of 30 volatile organic
compounds in whole human blood. The following compounds may be
determined by this method:
Compound CAS no.
Benzene 71-43-2
Bromodichloromethane 75-27-4
Bromoform 75-25-2
Carbon tetrachloride 56-23-5
Chlorobenzene 108-90-7
Chloroform 67-66-3
Dibromochloromethane 124-48-1
1,2-Oichlorobenzene 95-50-1
1,3-Dichlorobenzene 541-73-1
1,4-Dichlorobenzene 106-46-7
1,1-Oichloroethane , 75-34-3
1,2-Dichloroethane 107-06-2
1,1-Oichloroethene 75-35-4
trans-l,2-Dichloroethene . 156-60-5
1,2-Dichloropropane 78-87-5
cis-l,3-Dichloropropene • 10061-01-5
trans-l,3-Dichloropropene , 10061-02-6
Ethyl benzene • 100-41-4
Dichloromethane 75-09-2
Styrene 100-42-5
1,1,2,2-Tetrachloroethane 79-34-5
Tetrachloroethene 127-18-4
Toluene 108-88-3
1,1,1-Trichloroethane 71-55-6
1,1,2-Trichloroethane 79-00-5
Trichloroethene 79-01-6
Trichlorof1uoromethane 75-69-4
1,2-Xylene 95-47-6
1,3-Xylene . 108-38-3
1,4-Xylene 106-42-3
1.2 This is a heated dynamic headspace purge and trap gas chromato-
• graphic method using mass spectrometry in the limited mass scan
•monitoring mode. The method is applicable to the determination of
the compounds listed above in an approximate concentration range of
50 parts per trillion (ppt) to 5 parts per billion (ppb) in 35-mL
of whole human blood.
1.3 Quantisation is achieved by isotope dilution, where possible, or
versus an appropriate internal standard with purging characteristics
similar to the native compound.
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1.4 At this time, the estimated method detection limit (MDL) or limit
of detection (LOD) for each analyte has not been fully evaluated.
Preliminary investigations have determined the compound LODs to
range from 50 to 300 ppt in a 35-mL blood sample, depending upon
the compound.
2.0 Summary
2.1 Volatile organic compounds in whole blood are determined by a heated
dynamic headspace purge and trap GC/MS method. Stable isotopically
labeled analogs of compounds of interest are added to a 35-mL blood/
10-mL water sample contained in a specially designed purge vessel.
Prepurified nitrogen is passed over the surface of the heated (50°C)
and stirred blood/water mixture, removing volatile organics from
the sample into the gas stream. The purged sample components pass
through a two-way, six-port valve and are adsorbed on a polymeric
trap. After a known volume of purge gas has passed over the sample,
the adsorbent trap is heated and backflushed with prepurified helium,
desorbing the purgeable components onto the head of a wide-bore
fused silica GC column interfaced to a mass spectrometer. The mass
spectrometer is operated in the limited mass scan (LMS) mode where
only selected ions are scanned. Quantitation of a detected analyte
is determined from specific ion responses from standards of the ana-
lytes and their corresponding labeled analogs. The responses of
the labeled compounds are used to correct the variability of the
analytical technique through the use of an isotope dilution calcu-
lation procedure (EPA 1985) or an internal standard calculation
procedure.
3.0 Interferences
3.1 Compounds that have similar chromatographic properties and charac-
teristic mass spectral ions as the target compounds listed in Sec-
tion 1.1 may interfere with the determinations. Different ions may
need to be monitored to analyze samples that contain interfering
compounds.
3.2 Impurities in the purge gas and organic compounds out-gassing from
the plumbing ahead of the trap may account for some of the contami-
nation problems. The analytical system must be demonstrated to be
free from contamination under the conditions of the analysis by
running laboratory reagent blanks as described in Section 9.1. The
use of non-TFE plastic tubing, non-TFE thread se_alants, or flow
controllers with rubber components in the purging device should be
avoided.
3.3 The water used to dilute the whole blood samples is of particular
concern. Due to the extremely low levels achieved by this method,
the water used for sample dilution must be shown to be acceptably
free of the compounds of interest.
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3.4 Preparation of volatile-free water and handling of the blood sam-
ples should be conducted in a laboratory room dedicated to this
purpose or in some other designated volatile-free area. All
organic solvents, with the exception of methanol, should be pro-
hibited from use or storage in this laboratory.
3.5 Contamination by carryover can occur whenever high level and low
level samples are sequentially analyzed. To reduce carryover, the
purge vessel must be changed between sample analyses. Whenever a
sample with a high concentration of volatile organics is encoun-
tered, it should be followed by an analysis of reagent water to
check for cross contamination. The trap and .other parts of the
system are also subject to contamination; therefore, frequent bake-
out and purging of the entire system may be required.
4.0 Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. From this view-
point, exposure to these chemicals must be reduced to the lowest
possible level by whatever means available. The laboratory is re-
sponsible for maintaining a current awareness file of OSHA regula-
tions regarding the safe handling of the chemicals specified in
this method. A'reference file" of material, data handling sheets
should also be made available to all personnel involved in the chem-
ical analysis. Additional references to laboratory'safety are
available and have been identified (NIOSH 1977, OSHA 1976, ACS 1979)
for the information of the analyst.
4.2 The following parameters covered by this method have been tenta-
tively classified as "known or suspected,, human or mammalian carcin-
ogens: benzene, carbon tetrachloride, chloroform, dichloromethane,
and 1,4-dichlorobenzene. Primary standards of these toxic compounds
should be prepared in a hood. A NIOSH/MESA approved toxic gas res-
pirator should be worn when the analyst handles high concentrations
of these toxic compounds.
4.3 Analytes detected in the blood samples by this method are at trace
levels with minimal exposure risks to laboratory personnel. How-
ever, the blood samples should be handled with adequate safety pre-
cautions due to unknown microbiological activity in the blood.
Biosafety Level (BSL) 2 procedures'(NIH 1984) should be followed
when handling blood samples during the compositing and analysis
procedures. These procedures include handling blood samples with
protective gloves in a hood with an average face velocity of 100
linear feet per minute. After a sample has been analyzed, it should
be decontaminated with a chemical disinfectant, autoclaved, and dis-
posed of as biological waste. Specific handling instructions are
given in Appendix A of this method.
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5.0 Apparatus and Equipment
5.1 Sample Collection Vial - Glass vial (17 x 60 mm, ~ 8.5 ml) with a
Teflon-lined screw cap. Special cleaning described in Section 7.1
is required prior to use. Detergent wash, rinse with tap and
distilled water, rinse with pesticide-grade methanol, and dry at
105°C in the designated volatile-free area before use. An approxi-
mate 5-mL graduation line is to be made on the vial.
5.2 Purge and Trap Device - The purge and trap device consists of a
dynamic headspace purging vessel, a stainless steel adsorbent trap,
and a desorber heating element. Figure 1 shows the arrangement of
these components.
5.2.1 A purge vessel such as that shown in Figure 2 is used in
the determination of volatile organic content in the blood.
A vessel volume of 60 ml as measured to the tip of the purge
inlet is required. Any glass vessel of similar design and
function is adequate for this analysis. The vessel is con-
nected to the purge gas supply and purge/desorb valve via
Teflon tubing to allow flexibility and chemical inertness.
5.2.2 The stainless steel trap (10 cm x 2.64 mm ID) is packed with
~ 100 mg of Tenax TA® (35/60 mesh) adsorbent. Alternatively,
the trap can be purchased commercially as the required trap
for the EPA "Method 624-for purgeables. However, the silica
gel in this trap adsorbs water which subsequently is passed
into the GC/MS system and can cause degeneration of the ion
source after repeated analyses. The ion source may need to
be cleaned or replaced more frequently if silica gel" is used
in the trap.
5.2.3 Gas flow through the trap is directed with a two-way, six-
port valve. All transfer lines are heated to 80°C.
5.2.4 The Tenax trap is contained in a desorption unit activated
by a temperature sensing and controlling device. The de-
sorber heats the trap from 0°C to 220°C in less than 1 min
and maintains the temperature within a 5°C range.
5.3 GC/MS System
5.3.1 The gas chromatographic system includes a 30 m x 0.53 mm ID
DB-624 fused silica column (J&W Scientific, Folsom, CA).
The recommended GC parameters are:
Temperature Program: 30°C (2-min initial hold) to 225°C
at 10°C/min (10-min final hold)
Column Flow: 20 mL/min He
Purge Flow: 35 mL/min N2
Equivalent GC parameters may be used.
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Temperature
Controller
Two - way
Six-Port
Valve
Stainless
Steel (1/16")
Heated
Transfer
Line
Union
Antiseptic
Solution
(or Class A Hood)
Water
Temperature
Controller
Teflon
Tubing
(1/8"
O.D.)
Outlet - to
Tenax Trap
Water Jacket
Teflon Stopper
24/40 Joint
J/4" O.D.
Purge Gas Inlet
Flow
•Controller
for N2
• • -r
t -"'""'
~L_ i"~^' ,. .
IT
-aa,
/
Magnetic
Stirrer
Return Water Line
Figure 1. Dynamic headspace purge and trap equipment.
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1/4" O.D.
Outlet -
to Tenax Trap
Water Jacket
Water (50°C) jn
Teflon Stopper
24/40 Joint
Blood/Water
Sample
1/4" O.D.
Purge Gas Inlet
, ^
Water Out
Stir Bar
Figure 2. Volatile organic purge vessel.
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5.3.2 The recommended MS parameters are:
Electron Energy: 70 eV
Electron Multiplier Voltage: -1950 V
Emission Current: 0.3 mA
Scan Parameters: Limited mass ranges given below:
Total Scan Time: 1.1 s
Interval no.
Time request
Low mass High mass (s)
1
2
3
4
5
6
7
8
9
10
11
49 .,515
60.518
74.522,
80.524
90.527
110.534
115.535
126.538
145.544
163.549
168.550
56.517
68.520
79.523
88.526
106.532
113.534
120.536
131. 540
152. 544
166.550
174.552
0.100
0.100
0.100
0.100
0.200
0.050
0.100
0.100
0.100
0..050
0.100
Equivalent MS parameters may be used with the exception of
the mass ranges. Different mass ranges may be used if
interferences are encountered in the analysis of selected
samples. Changing the mass range scanned, however, would
require recalibration of the GC/MS system.
5.3.3 A data system is interfaced to the mass spectrometer for
acquisition of a data file for each chromatographic analy-
sis. The raw data are stored on accessible discs for manual
interpretation. Ion abundances of a specific mass are
plotted versus scan number. The computer measured peak
areas of the characteristic mass fragmentation ions of a
particular analyte and its corresponding labeled analog are
used to determine the concentration of the analyte in the
blood.
5.4 Glassware - Fixed needle glass syringes with ± 1% accuracy are re-
quired for addition of internal standard to the solution. Syringes
of 10, 50, 100, and 250 uL should be available for dilution of stock
solution for standard preparation.
5.5 Analytical Balance - Analytical balance accurate to the nearest
0.1 mg (for standards preparation) and to the nearest O.Ol.g (for
sample weight determination).
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6.0 Reagents, Materials, and Standards
6.1 Methanol - Pesticide quality or equivalent for dilution of stock
standards and glassware preparation.
6.2 Reagent Water - The water used in the dilution of the blood sample
is obtained from a Millipore® reagent water purification system.
This water is then passed through a carbon filter bed into a glass
holding tower where the water is held at 50°C and continuously
purged with prepurified nitrogen. It is recommended that this
volatile-free water be generated and stored in the designated
volatile-free area.
6.3 Stock Standard Solution - Stock standard solutions can be prepared
from neat compounds of 99% purity on the target list (Section 1.1)
Neat materials can be obtained from commercial suppliers (e.g.,
Aldrich Chemical Company). Individual stock standards in a water
miscible solvent can be obtained from the EPA Repository for Toxic
and Hazardous Materials (EPA/EMSL-Cincinnati).
Stock standard solutions are prepared from neat compounds by deter-
mining the weight of the neat compound added to a known amount of
methanol. Specifically, place approximately 9.8 ml of methanol
into a 10-mL ground glass stoppered volumetric flask. Allow the
flask to stand unstoppered for about 10 min or until all alcohol-
wetted surfaces have dried. Weigh the-flask to the nearest 0.1 mg.
.Then, using a 100-uL glass syringe, add two or more drops of neat
compound to the flask and reweigh. Alternatively, using the den-
sity of the compound, determine the volume needed for a specific
mass, deliver that amount to the flask, then reweigh the flask.
In either case, the difference in weight is the amount of neat
compound added.
The liquid must fall directly into the solution without contacting
the neck of the flask. After the weight of the added compound has
been determined, dilute the standard to volume with methanol, stop-
per, and invert several times before diluting to secondary standard
levels.
The stock standard solutions are stored at -20°C in Teflon-lined
screw-cap vials. The recommended maximum storage time for'concen-
trated stocks in methanol is 1 month under these conditions.
6.4 Secondary Dilution Standards - Mixed secondary standards are pre-
pared by diluting the stock standard solutions into one solution of
methanol. Dilutions of this mixed secondary standard are made to
generate working solutions for the calibration curve. These working
solutions should be freshly prepared on a weekly basis. Secondary
standards can be stored at -20°C for up to 2 weeks.
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6.5 Labeled Analog Solution - Stock solutions of the labeled analogs
are prepared from the neat compounds following the same procedures
as described in Section 6.3. Neat materials can be obtained from
commercial suppliers (e.g., MSD Isotopes). A mixed analog spiking
solution is prepared by taking an aliquot of each stock solution to
an exact volume of methanol. Dilutions of this solution are made'
to yield an analog concentration of 3.5 ng/uL for each of the labeled
compounds. With each sample analysis, 5 uL of this solution is added
to the purge vessel. Stock solutions can be kept for 1 month at
-20°C. Working solutions should be freshly prepared on a weekly
basis.
6.6 Mixed Analyte/Analoq Solutions - Aliquots of the mixed secondary
analyte solution (Section 6.4) and the mixed labeled analog solution
(Section 6.5) are combined to generate working solutions for the
calibration curve. These solutions are prepared such that the con-
centration of the labeled analogs remains constant and the analyte
concentrations are varied. These calibration solutions should be
kept at -20°C for storage and should be freshly prepared on a weekly
bas is.
6.7 Anticoagulant Solutions - An aqueous anticoagulant solution is pre-
pared by dissolving 500 mg of EDTA (ethylenediaminetetraacetic acid
disodium salt, Fisher Scientific Company) in 10 ml of organic-free
reagent water for a final concentration of 50 mg/mL Two hundred
. • microliters of this solution is used for each 5 ml of blood col-
lected. This solution should be freshly prepared on a monthly basis.
6.8 Charcoal - Activated charcoal (cocoanut, activated, 8-12 mesh) is
used for the storage of samples.
6.9 Trap Materials - 2,6-Diphenyloxide polymer - Tenax® TA (35/60 mesh).
6.10 Disinfectant Solutions - Household bleach containing at least 5%
NaOCl (e.g., Clorox®) diluted 1:10 with tap water.
7.0 Sample Preservation, Collection, and Storage
7.1 Sample Preservation - Prior to collection of the blood samples, the
sample vials are rinsed with reagent water and pesticide quality
methanol. The vials should then be dried at least 4 h at 105°C.
After the vials have cooled in a volatile-free environment (e.g.,
sealed chamber containing charcoal), 200 uL of the EDTA anticoagu-
lant solution at 50 mg/mL is added to each vial. _ The vials are'then
capped with Teflon-lined screw caps and stored fn containers with
charcoal in the designated volatile-free area until used for blood
collection.
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7.2 Collection - The blood sample is collected by the blood center per-
sonnel after the collection of the donated unit of blood and any
pilot tubes. To collect the blood sample, disconnect the collec-
tion bag from the blood collection tubing by clamping the tubing to
stop blood flow. Then cut the tubing between the clamp.and the
blood bag, process the unit as normal, and fill any pilot tubes as
necessary. Open the two collection vials. Unclamp the tubing and
allow the blood to flow from the donor through the tubing into two
collection vials. Fill each vial to the 5-mL graduated mark. Cap
the vials. Remove the needle and tubing from the donor. Invert
the vials at least 10 times to mix the anticoagulant with the blood
sample.
7.3 Storage - Following collection of the blood sample at the blood col-
lection center, the sample is transferred to the shipping container
as soon as possible. The shipping container will be fitted with
vial racks, which will hold the samples. Wet ice will be added to
cool the samples to about 5 to 10°C. After receipt and log-in at
the analytical laboratory, the samples should be grouped according
to the sample-compositing design and placed in a wide-mouth jar
half-filled with cocoanut charcoal. The jar should be sealed and
placed in a refrigerator at 4°C. The blood samples for volatile
analysis should never be frozen.
8.0 Calibration
Mass calibration of the mass spectrometer is conducted according to manu-
facturer specifications. The isotope dilution method is based upon the
response and chemical behavior relationship between the analyte and its
labeled analog. Method calibration of the GC/MS system can be conducted
by direct injection of varying levels of the analytes with a constant
amount of the corresponding labeled analog. That is, standards need not
be purged from water to calibrate the purge and trap-GC/MS system.
8.1 The gas chromatograph must be operated using temperature and flow
rate parameters equivalent to those in Section 5.3.1.
8.2 Mass calibrate the GC/MS system daily with an instrument manufac-
turer's specified calibration gas (e.g., perfluorotributylamine) to
ensure proper mass identification. Since this is a low resolution
method, only unit resolution is required.
8.3 Calibrate the GC/MS system using isotopic dilution and internal
standard techniques as described below.
8.3.1 Mixed analyte/analog working solutions (Section 6.6) are
used to generate a calibration curve for each analyte in
the range of 2 ng to 200 ng. This corresponds to ~ 60-ppt
to ~ 6-ppb levels for a 35-mL (35-g) composited blood sam-
ple.
10
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Analyze the standards by direct injection into the GC/MS
system. Additional higher and lower level standards may
also be analyzed until nondetection or nonlinearity occurs.
8.3.2 Keep the labeled analog amount in each injection constant.
An amount of 17.5 ng (equivalent to 500 ppt) is recommended.
8.3.3 The analyte concentration range should consist of at least
five separate concentration levels (e.g., 2-10-50-100-200 ng).
•8.3.4 If analytes are detected in the blood/water mixture at levels
greater than 200 ng, the calibration curve is extended to
bracket those analytes, if possible. If the extended cali-
bration curve becomes nonlinear, then a duplicate aliquot
of the original blood sample is diluted and reanalyzed within
the range of the established calibration curve. If no dup-
licate is available, the concentration of the analyte is
qualified by indicating it is outside the calibration curve
(e.g., > x, where x is the highest level of the analyte
standard).
8.4 Isotope Dilution Calculations - The labeled analogs chosen for quan-
titation of the standard analytes in the blood matrix are shown in-
Table 1. A labeled analog, deuterium (d) or carbon-13 (13C), is
used to quantitate the corresponding analyte by-the isotope dilution
method. The amount of labeled isotope remains constant during cali-
bration 'and sample analysis. The amount of analyte is varied from
2 to 2.00 ng to generate relative response factors. The isotope
dilution method is different from the internal standard method only
in cases where there is contribution to the response of the analyte
quantitation ion by the analog, or vice versa. In these cases, the
calculations explained in the following section are used to'determine
the RRF (relative response factor) for the analyte.
8.4.1 The relative response of the quantitation ion of the analyte
(Table 2) to the quantitation ion of its labeled analog
(Table 3) is determined from isotope ratio values computed
from GC/MS analyses of the analytes only, the analogs only,
and a mixture of analytes and analogs. Three isotope ratios
are determined: Rx = the isotope ratio measured for the
pure analyte; Ry = the isotope ratio measured for the pure
labeled compound; and Rm = the isotope ratio of the analyt-
ical mixture of analyte and labeled compounds (as in the
calibration curve solutions). Quantitation ions have been
selected so that Rx > Ry and Rm is between 2Ry and O.SRx.
8.4.1.1 To begin calibration of the GC/MS system, analyze
a solution containing only the analytes at a known
concentration (e.g., 20 ng). Then analyze a solu-
tion of only the labeled analogs at the same con-
centration (e.g., 20 ng). From these data, the Rx
and Ry isotope ratios are determined as given below.
11
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Table 1. Target Compounds and Corresponding Labeled Analogs
Compounds
Labeled analog
Benzene
Bromodichloromethane
Bromoform
Carbon tetrachloride
Chlorobenzene'
Chloroform
Dibromochloromethane
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
1,1-Dichloroethane
1,2-Oi ch1oroethane
trans-1,2-Dichloroethene
1,2-Oichloropropane
cis-l.S-Oichloropropene
trans-1,3-Oichloropropene
Ethyl benzene
Styrene
1,1,2,2-Tetrach1oroethane
Tetrachloroethene
Toluene
1,1,1-Tri chloroethane
1,1,2-Trichloroethane
Tri chloroethene
1,2-Xylene
l,3-Xylene/l,4-Xylene
Dichloromethane >c
1,1-Dichloroethane0
Trichlorof1uoromethane
Benzene-d6
l,2-Dichloropropane-d6
Bromoform-13C
Carbon tetrachloride-13C
Chlorobenzene-d5
Chloroform-d
Chlorobenzene-dsa
l,3-Dichlorobenzene-d4
1,3-Dichlorobenzene-d4
l,3-Dichlorobenzene-d4
•l,l-Dichloroethane-d3
1,2-0ichloroethane-d4
trans-1,2-Di chloroethene-d?.
1,2-Di cnloropropane-dg
c_j_s-l,3-Dichloropropene-d4
trans-1,3-D ichloropropene-dd.
Ethylbenzene-d10
S.tyrene-dg
1,1,2,2-Tetrach1oroethane-d2
Tetrachloroethene-13C2
Toluene-dg
1,1,1-Tri chloroethane-d3
1,1,2-Tri chloroethane-ds
Trichloroethene-13C
l,2-Xylene-d10
l,2-Xylene-d10
Dichloromethane-d2
b$uggested internal standard.
High levels of native dichloromethane contribute significantly to dichloro-
Cmethane-d2 and may preclude quantisation.
These compounds did not meet the accuracy and precision criteria established
for validation of the method (50-150% recovery). These compounds should be
reported as qualitatively detected.
12
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Table 2. Quantitation and Confirmation Ions for Target Compounds
Compound
Benzene
Bromodichloromethane
Bromoform
Carbon tetrachloride
Chlorobenzene
Chloroform
Dibromochloromethane
1, 2- Di chlorobenzene
1, 3-D i chlorobenzene
1,4-Di chlorobenzene
1 , 1-Di chl oroethane
1,2-Di chl oroethane
1,1-Dichloroethene
trans-l,2-Di chl oroethene
Dichloromethane
1, 2-Di chl oropropane
cis-l,3-Dichloropropene
trans- 1,3-Dichloropropene
Ethyl benzene
Styrene
1 , 1 , 2 , 2-Tetrach 1 oroethane
Tetrach 1 oroethene
Toluene
1,1, 1-Tri chl oroethane
1,1,2-Tri chl oroethane
Tri chl oroethene
Trichlorofluoromethane
1,2-Xylene
1,3-Xylene
1,4-Xylene
Quantitation
ion
78
83
173
117
112
83
129
146
146
146
65
64
96
96.
84
76
75
75
9.1
104
83
164
91 .
97
97
130
101
91
91
91
Confirmation
ion
so :
85
171
119
77
85
127
148
148
148
63
62
61
61
86
62
77
77
106
103'
85
129
92
99
83
95
103
106
106
106
13
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Table 3. Quantitation and Confirmation Ions for Labeled Analogs
Compound
Quantitation
ion
Confirmation
ion
Benzene-d6
Bromoform-13C
Carbon tetrach1oride-13C
Chi orobenzene-ds
Chloroform-d
l,3-Dichlorobenzene-d4
l,l-Dichloroethane-d3
l,2-Dichloroethane-d4
trans-1, 2-Dich1oroethene-d2
Dichloromethane-d2
l,2-Dichloropropane-d6
cj_s-l,3-Dichloropropene-d4
trans-1 ,3-Di chl oropropene-d4
Ethyl benzene-d10
Styrene-d8
1,1,2, 2-Tetrachl oroethane- d2
Tetrachl oroethene- 13C2
Toluene-d8
1,1, 1-Tri chl oroethane-d3
l,l;2-Trichloroethane-d3
Trichloroethene-13C
l,2-Xylene-d10
84
172
118
117
86
152
68
67
65
88
67
79
79
98
112
86
172
98
100
102
99
116
56
174
120
119
84
150
66
65
100
51
81
81
81
116
84
84
170
100
102
100
,101
98
14
-------
For Rx,
n _ area quantitation ion of analyte
area analyte contribution to the quantitation
ion of labeled analog
at the retention time (RT) of the analyte during
the analyte-only analysis. For Ry,
Ry =
area labeled analog contribution to the
quantitation ion of analyte
area quantitation ion of labeled analog
at the retention time of the labeled analog during
the labeled analog-only analysis.
If no area is detected for a given ion as deter-
mined by the qualitative criteria given in Section
11.0 (i.e., there is no contribution), a value of 1
is assigned in the equations given above.
8.4.1.2 To determine the value of Rm and subsequently RR
(relative response), analyze a 1.0-uL aliquot of
each calibration standard (Section 8.3). Rm is
calculated using the following equation:
Rm = area quantitation ion of analyte at RT of analyte
area quantitation ion of analog at RT of analog
Rm is then used to determine the RR (relative
response) of the mixture in each of the three
cases given in the following sections.
8.4.1.2.1 If the analyte-analog pair is chromato-
graphically separated such that the
height of the valley between the two
peaks at the same m/z is less than 10%
of the height of the shorter of the two
peaks, then the relative response (RR) =
Rm and no correction is needed for over-
lap contributions.
8.4.1.2.2 If there is no mass contribution to the
labeled quantitation ion from the Rx
determination (Section 8.4.1.1) and
there is no contribution to the analyte
quantitation ion from the Ry determina-
tion (Section 8.4.1.1), then the relative
response (RR) = Rm.
8.4.1.2.3 If the analyte-analog pair is not chro-
matographically separated and if respec-
tive mass contributions were found from
15
-------
Rx and Ry determinations (Section 8.4.1.1),
then the relative response (RR) is calcu-
lated using the following equation:
RR _ (Ry - Rm)(Rx + 1)
KK (Rm - Rx)(Ry + 1)
where Rx and Ry are values determined
in Section 8.4.1.1 and Rm is the analyte/
analog peak area ratio in the mixture
being analyzed (Section 8.4.1.2). Rm
must be between 2Ry and 0.5Rx (2Ry < Rm
< .5Rx). The calibration curve becomes
nonlinear at the point where Rm falls
outside this limit. Significant mass
contribution has been observed for
dichloromethane and trichloroethene.
Alternatively, the analyte could be
quantitated by the internal standard
method relative to a different labeled
analog (Section 8.5).
8.4.2 After the relative response (RR) has been determined (Sec-
tion 8.4.1.2), the relative response factor (RRF) is then
calculated as follows.
DOC - DD ~ ng of labeled compound
t\l\r — KK X — ,- -i .
ng of analyte
for each level of the analyte in the calibration curve.
8.4.3 Consider the following example for clarification of Sections
8.4.1 and 8.4.2 calculations.
• Analyze 1.0 uL of the 20 ng/uL solution containing only
the anaTytes ' (analyte response = 10,000 area units;
contribution to the labeled analog response = 1,000 area
units).
_ 10,000 _ ,n
- 10
Analyze 1.0 uL of the 20 ng/uL solution containing only
the labeled analogs (contribution to the analyte response
= 500 area units; labeled analog response = 15,000 area
units).
Analyze a 1.0-uL calibration standard containing 100 ng/|jL
analytes and 20 ng/uL labeled analogs. The analyte-analog
16
-------
. pair is not chromatographically separated (analyte
response =40,000 area units; labeled analog response =
10,000 area units).
Rm = 40,000 = 4
Km 10,000 4
' RR = (Ry - Rm)(Rx + 1)
(Rm - Rx)(Ry + 1)
_ (0.03 - 4)(10 + 1)
(4 - 10)(0.03 + 1)
RR = 7.1
• RRF _ RR x ng labeled analog
ng analyte
= 7.1 x 20 ng/100 ng
RRF = 1.4
8.5 Internal Standard Calculations - The internal standard method is
used to quantitate those compounds where a corresponding labeled
analog of the compound is not available. The internal standards
recommended for quantisation of the analytes in the blood matrix
are shown in Table 1, The area responses of the quantitation ion
for each analyte relative to the area response of the quantitation
ion for the specified internal standard are tabulated to determine
a relative response factor (RRF). The amount of internal standard
remains constant. The amount of standard will be varied from 2 to
200 ng to generate relative response factors using' the equation below.
AS x ng IS
RRF = AJS x ng S
- where AS = area of the quantitation ion for the analyte measured;
ng IS = total nanograms of the specified internal standard in-
jected into the GC/MS system;
AT_ = area of the quantitation ion for the internal standard;
and Xi
ng S = total nanograms of the analyte injected into the GC/MS
system.
8.6 Ideally, the relative response factors are constant over .the entire
concentration range of interest. If the RRF value is constant over
the working range (^ 30% RSD), the average RRF will be. used for
determining the concentration of a given analyte in a blood sample
(Section 13.0). If not, analyte concentrations must be determined
from the actual calibration curve by plotting RR versus analyte
concentration for those compounds determined by isotope dilution
(Section 8.4). For those compounds quantitated by the internal
17
-------
standard method (Section 8.5), the concentration must be determined
by plotting analyte/analog response versus analyte concentration.
8.7 A dally check of the instrument sensitivity will be performed at
the beginning and end of each day's analysis with a low level and
mid level calibration standard, respectively. The daily relative
response factor (Sections 8.4 and 8.5) for each analyte must be
± 30% of the cumulative average relative response factor from the
calibration curve and succeeding standard analyses. The daily
relative response factor is then incorporated into the average
relative response factor from the calibration curve, and a cumu-
lative response factor average is used to quantitate each day's
analysis.
If the daily response factor is outside the 30% limit, the daily
standard analysis must be repeated using a fresh calibration
standard. If the criterion is still not met, a new calibration
curve and relative response factors must be established.
8.8 External Standard Calibration (Analogs Only) - The external standard
method is used to establish a calibration curve for determining
percent recovery of the labeled analogs from the blood matrix. The
recoveries of the added analogs are used to determine the stability
of the system (Section 9.5). Dilutions of the labeled analog solu-
tion (Section 6.5) are made to yield standards corresponding to
20, 50, and 100% recovery. The. standards are analyzed by direct
injection (e.g., 3.5, 8.75, 17.5 ng) to form a three-point analog
calibration curve of area units versus percent recovery. Percent
recovery of the labeled analog in a blood analysis is determined
directly from the linear regression curve. These standards are
analyzed throughout the day to monitor changes in instrument sensi-
tivity. The suggested sequence of analysis is given in Table 4.
9-0 Quality Control
9.1 A reagent blank consisting of volatile-free water and labeled ana-
logs will be analyzed with each day's sample set. Forty-five mini-
liters of reagent water is added-to a purge vessel with 17.5 ng of
each labeled analog and analyzed as an actual sample. Contaminants
are determined and quantitated relative to their corresponding
labeled analog.
9.1.1 Reagent water (Section 6.2) is required for dilution of the
blood samples. A sufficient volume to complete the day's
analyses is collected from the holding tower (Section 6.2)
and transferred to a precleaned glass container. This
entire volume of water is prepurged again at the site of
analysis (GC/MS laboratory) and then, transferred 'back to
the precleaned glass container. This water is used for
dilution of the blood during the day's analyses, and for
the reagent water blank.
18
-------
9.1.2 A water blank should be analyzed at the beginning of each
day's analysis, after the daily standard, to determine
potential contamination from diluting the samples with water.
If unacceptable levels of target compounds are found in the
reagent water (e.g., > 50 ppt), the analysis is repeated.
If contamination still exists, new reagent water should be
prepared.
9.1.3 Field blanks consisting of EDTA and reagent water in a col-
lection vial will be shipped to and 'from a collection site
with the field samples and will be analyzed for each set of
samples from a site.
9.2 After generation of the calibration curve, a calibration standard
should be analyzed daily to ensure reproducibility. A low level
standard is run at the beginning of the day, and a mid level stan-
dard is run at the end of the day. The relative response factors
for each analyte should be checked against the average relative
response factor from the established calibration curve. Agreement
should be within 30% of the established RRFs. If they are not, a
new standard is prepared and analyzed or a new calibration completed.
9.3 A duplicate sample will be analyzed for each 10 samples analyzed.
If there are less than 10 samples in a specific batch, at least one
duplicate will be analyzed. Sample collection includes collecting
two vials of blood (Section 7.2). For duplicate analyses, the second
vial collected will be used to form the duplicate composite sample.
Half of the duplicates will be analyzed on different analysis days
than the corresponding original so that between day precision can
be determined.
9.4 A spiked blood sample will be prepared from duplicate samples col-
lected and will be analyzed for each 10 samples analyzed. If there
are less than 10 samples in a specific batch, at least one spiked
sample will be analyzed.
9.5. Since labeled analogs are spiked into each blood sample, recovery
of these compounds can be calculated versus their response' in direct
-injection by the external standard method. Initially, a default
control limit of ± 30% from the average absolute recovery will be
used. After a database of labeled analog recoveries (R) has been
established and upper and lower control limits defined based on the
standard deviation of the recoveries, these criteria can be used to
determine whether or not a sample needs to be reanalyzed. Initially,
if 70% of the compound recoveries are in control-; analyses can con-
tinue, otherwise the analysis must stop until the problem is cor-
rected. The upper and lower control limits are calculated using
the formula:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R = average labeled analog recovery
s = standard deviation of recoveries
19
-------
It is recommended that the UCL and LCL "limits based on a 40-point
"float" technique. The UCL and LCL criteria are first established
after 20 analyses. These control limits should be used to evaluate
the results from the next 20 analyses. New UCL and LCL criteria
are then calculated for the first 40 data points and are used to
consider the quality of the next 20 QC analyses. The first 20 data
points are dropped and the final 20 data points are included to
determine the 40-point "float" average.
9.6 External Quality Controls Checks - Performance audit standards will
be prepared by an external quality control coordinator (QCC). The
purpose of this standard is to check the performance of the instru-
ment and the accuracy of the calibration curve. The concentration
of the standard must be in the working range of the calibration curve.
Labeled analogs must be included in this solution at the same concen-
tration as in the calibration standards. The performance audit
standard will be analyzed by direct injection. Total nanograms of
analytes detected will be calculated according to the calibration
established in Section 8 and the results reported to the QCC. Ac-
curacy will be determined by the QCC. If the accuracy is 70 to 130%,
the performance objectives will be considered acceptable. If the
limits of accuracy are not reached, another performance standard is
analyzed. If the limits of accuracy are still not reached, a new
calibration curve will be generated from freshly prepared standards.
Table 4 shows the sequential analyses on a. typical day.
10.0 Procedure
10.1 This analysis procedure covers the analysis of single samples
(~ 5 mL) and composited (seven 5-mL samples) samples.
10.1.1 Prior to the analysis of each sample, whether analyzed
singularly or in a composite, determine the combined weight
of the sample and the collection vial. After transference
of the sample(s) to the purge vessel without rinsing, the
empty vial will be reweighed and weight of blood determined
and recorded.
10.1.2 Single Sample Method - Place 40 mL of organic-free reagent
water in the purge vessel. Add a Teflon-coated stir bar to
the vessel. Transfer the blood sample (approximately 5 mL;
actual weight will be determined as described in Section
10.1.1) to the purge vessel in a hood. Immediately spike
the blood/water mixture with 5 uL of the-'labeled analog
(3.5 ng/uL) working solution (Section 6.5). Close the
vessel and connect the vessel to purge and trap system.
Recap the sample vial for weight determination and subse-
quent disposal by autoclaving.
20
-------
Table 4. Scheme for Typical Day's Analysis
1. Mass calibrate the GC/MS system (Section 8.2).
2. Inject a low level mixed standard. Check response factors (Section 8.7),
3. Inject an analog-only standard (Section 8.8).
4. Analyze 45 ml of the reagent water (Sections 9.1.1 and 9.1.2).
5. Analyze blood samples (Section 10.0).
6. Analyze a second level analog-only standard (Section 8.8). Check for
sensitivity changes in the instrument.
7. Analyze blood samples (Section 10.0).
8. Analyze the third level analog-only standard (Section 8.8).
9. Analyze a mid-level mixed standard (Section 8.7).
21
-------
10.1.3 Composite Sample Method - The seven blood samples are com-
posited in a hood and in an environment that is as volatile-
free as possible. This is done in close proximity to the
GC/MS instrument to minimize contamination during transpor-
tation of the sample to the instrument. To composite the
samples, place 10 ml of volatile-free water in a clean purge
vessel with a Teflon stirring bar. Immediately add, with
no stirring, each of the individual blood samples to be
composited to the vessel (approximately 5 ml each; actual
weight will be determined as described in Section 10.1.1).
Immediately spike the blood/water mixture with 5 uL of the
labeled analog (3.5 ng/uL) working solution (Section 6.5).
Quickly close the vessel and connect it to the purge and
trap system without delay. Recap the sample vials for
weight determination and disposal after autoclaving.
10.1.4 A constant temperature recirculating water bath is equili-
brated at 50 ± 5°C. After the blood has been transferred
to the purge vessel, immediately connect the recirculating
bath hoses to the jacketed purge vessel. Begin stirring
and purge the sample as directed in Section 10.2.
10.2 Purge - With the adsorbent trap at ice water temperature (0°C),
ensure that the valve is in the purge position, begin the stirring
of the blood/water solution, start the purge flow, and purge the
sample for 20 min. Check the purge flow to ensure "a 35 ± 2 mL/m.i.n
gas flow is being maintained.
10.3 Desorption and Data Acquisition - After the 20-min purge to the trap
is complete, turn the valve to the desorb mode, activate the trap
. temperature controller to heat the trap, begin the GC temperature
program, and begin the GC/MS data aquisition. Leave the trap at
220 ± 5°C and the valve in the desorb mode until the GC temperature
program and data acquisition are complete. This procedure will
ensure that the trap and tubing are cleaned between samples and
carryover is minimized.
10.4 Vessel Change - During the desorption and data acquisition, discon-
nect the purge vessel from the system. Connect a clean vessel to
the system and allow the purge flow to pass through the vessel to
the transfer lines and out the vent line to clean the system and
minimize carryover between samples. When data acquisition from the
previous analysis is complete, cool the trap to 0°C. Prepare the
next sample for analysis as described in Section 10.1.2 or Section
10.1.3. Dispose of the sample that has been analyzed into a plastic
gallon jug containing Clorox® (1:10 dilution), "immerse the used
purge'vessel in a Clorox® bath (1:10 dilution) for 5 min and wash
the vessel with.detergent, rinse with water and pesticide quality
methanol, then water, and dry in an oven at 105°C.
22
-------
11.0 Qualitative Identification
11.1 Obtain the extracted ion plots (EICPs) for the quantisation and
confirmation ions for each compound of interest and its labeled
analog (Tables 2 and 3). The following criteria must be met to
make a qualitative identification.
11.1.1 The characteristic ions (quantisation and confirmation) of
each compound of interest must maximize in the same or
within one scan of each other.
11.1.2 The retention time of the analyte must fall within ± 5 scans
of the retention time of the corresponding labeled analog.
11.1.3 The peak area ratio of the two characteristic ions in the
EICPs must fall within ± 30% of the peak area ratio of these
ions in the daily calibration standard.
11.1.4 The quantitation ion must consist of at least two chromato-
graphic data points.
Figure 3 shows the qualitative identification procedure in flowchart
form.
11.2 Structural isomers that have very similar mass spectra and less than
30 s difference in retention time can be explicitly identified only
if the resolution between authentic isomers in a standard mix is
, acceptable. Acceptable resolution is achieved if the baseline to
valley height between the isomers is less than 25% of the sum of
the two peak heights. Otherwise, structural isomers are. identified
as isomeric pairs.
11.3 After qualitative identification of an analyte, determine its con-
centration in the blood according to the procedures in Section 13.0.
12.0 Compounds to be Reported as Qualitatively Identified
Three of the 30 analytes included in this method are reported on a quali-
tative basis. These compounds are 1,1-dichloroethene, dichloromethane,
and trichlorofluoromethane. Preliminary spiked recovery data for these
compounds were outside the accuracy range established for the method
validation work (Appendix B, Table B-l).
The data for these compounds will be qualified as qualitatively detected
(QD) in the data reporting form (Section 16.0).
13.0 Calculations
13.1 Labeled analogs are added to each sample in the purge vessel. The
amount of each labeled analog added is 17.5 ng (5 uL of a mixed
solution containing 3.5 ng/uL of each labeled analog).
23
-------
Qualitative
Identification of
Volatile Organic:
in Blood
1
f
Examine EICP for
Analyte and Analog
- Determine RT of
Analog
Analyte
Quant. Ion
within 5 Scans
of Analog ?
Analyte Not
Detected - Report
MDLas Calculated
from Daily Spika
Analyte
Quant. Ion
Consist of at
Least Two
Chrom. Data
Points ?
Analyte
Characteristic
Ions Maximize
within one
Scan?
Analyte Not
Detected - Calculate
MDLas if Detected
Compound were
Analyte
Characteristic
Ion Ratios within
£30% of Daily
Standard?
YES
Analyte Detected-
Quantitate as per
Protocol
Figure 3. Qualitative identification procedure.
24
-------
13.2 After a peak has been qualitatively identified, quantisation will
be performed relative to the appropriate labeled analog shown in
Table 1. The area of the quantisation ion for a given compound will
be used for calculations (Table 2).
13.3 The total amount of analyte present in the sample is determined using
the average relative response factor for that analyte from the up-
dated calibration curve (Section 8.7). Use the same ions for quan-
ti tat ion of the samples and standards as given in Tables 2 and 3.
13.3.1 For the isotope dilution calculation:
Total analyte (ng) = RR x amount 1abgjgd ana1°9 (nc°
RRF
where RR = relative response of analyte to labeled analog,
where Rm and subsequent RR values for the
analyte in the sample are determined from
the equations given in Section 8.4.1.2; and
RRF = average relative response factor for the analyte
versus the 'labeled analog as determined from
the updated calibration curve (Section 8.7).
13.3.2 For the internal standard method, when a corresponding
labeled analog is not available for isotope dilution, the
following formula is used to determine the amount of an
analyte in a blood sample:
A- x ng IS
Total analyte (ng) = — --
A,.- x RRF
where AS = area of the quantisation ion for the compound
to be measured;
ng IS = total nanograms of the internal standard added
to the sample;
A~ = area of the quantisation ion for the
internal standard; and
RRF = average relative response factor for the
compound versus the internal standard as
determined! from the updated calibration
curve (Section 8.7).
Figure 4 shows the procedure for quantitating an analyte in
flow chart form. In the flow chart calculated levels of
analytes greater than or equal to three times the historical
MDL for that analyte (Section 14.0) are labeled as "positive
quantifiable." Analytes with calculated levels that are
less than three times the MDL but greater than the historical
MDL are labeled as "trace." If the calculated analyte level
does not meet the qualitative criteria (Section 11.0), the
analyte is labeled "not detected."
25
-------
Analyte
Quantitation
Procedure
Response
Meets Al
Qualitative
Criteria?
Report as ND
-Calculate MDL
Calculate Analyte
Level as per Protocol
Analyte
Level Less
than 3x
Label as Positive
Quantifiable
Figure 4. Analyte quantitation procedure.
26
-------
13.4 After the total nanograms of an analyte from the blood sample are
determined, the weight of the blood sample as determined in Section
10 will be used to calculate the concentration of the analyte in
the blood in ng/g using the equation below.
Total ng analyte , ,.,..,,
g blood - g anticoagulant = ng/g analyte ™ blood.
13.5 Corrections must be made for any contaminants from the daily water
blank (Section 9.1.1). The area response of the quantisation ion
for a detected analyte in the blank will be adjusted by the appro-
priate labeled analog (Table 1) response. Total nanograms of ana-
lyte detected in the water blank (45 mL) will be determined from
Section 13.3 calculations. The total nanograms of analyte per
milliliter of water will then be determined (total analyte detected/
45 ml = ng/mL). This ng/mL analyte concentration will be used to
determine total nanograms of analyte in the 10 mL of water used to
dilute the blood (total ng = 10 ml. x ng/mL). Total nanograms deter-
mined in the 35-mL blood/10 mL water mixture will be adjusted by
subtraction of the amount of analyte attributed to the 10 mL of
water in the mixture.
13.6 Field blank values will be reported with the corresponding field
sample results.
14.0 Precision and Accuracy
14.1 Precision - Duplicate samples analyzed in Section 9.3 can be used
to calculate a range percent, P (%) , to estimate the precision of
the method using the equation:
x 10o
where C^ = high concentration of given analyte in sample;
C2 = low concentration of given analyte in corresponding
_ duplicate sample; and
C = average concentration of analyte.
14.2 Accuracy - The accuracy of the method can be assessed from the analy-
sis of spikes generated in Section 9.4. The measurement for percent
accuracy, A (%), will be:
A (0,-v _ Spiked sample value - Unspiked sample value
Amount of spike x 10°
14.3 A preliminary evaluation of the precision and accuracy of this method
has been performed from a 4 level x 4 replicate study. The accuracy
and precision results of this study are given in Appendix B, Table B-l.
Selected analytes were chosen for additional evaluation in a 3 level x
3 replicate study. The results of this study are presented in Appen-
dix B, Table B-2.
27
-------
15.0 Estimated Method Detection Limit (MDL)
The estimated MDLs given in Appendix B, Table B-l, were determined from
spiked sample data and are based on 35-mL samples. These MDLs may be
initially adopted by the analyst but are subject to change as the data-
base of spiked sample information increases. Initially, the analyst must
demonstrate the ability to detect these levels in whole, blood by perform-
ing duplicate analyses of blood samples spiked at the estimated MDLs for
each compound. The analytes must meet the qualitative criteria set forth
in Section 11.0. If 70% of the analytes are not detected in both of the
spiked samples, the analyst must increase the GC/MS sensitivity (e.g., by
increasing the EM voltage of the MS) until the MDLs given in Appendix 8,
Table B-l, are achieved. If the estimated MDLs in Table B-l are not
achieved, the EPA Project Officer must be notified of the higher operat-
ing MDLs. If lower MDLs are achieved than those specified in this method,
then new MDLs are established by the method given in Section 15.1.1 or
by using a scientifically acceptable statistical method (Long 1985,
Keith 1983).
There are several methods currently in use to determine estimated method
detection limits. Among these are determinations from noise levels, from
regression analyses, and from spike sample data. The method presented
in Section 15.1.1 determines MDLs from the daily sample spikes.
15.1 Esti-mated method detection limits can be calculated In'situations ."
where (1) no response is observed for an analyte; (2) a response is
observed but ion ratios are incorrect; and (3) where a response is
quantitated as a trace value (i.e., where the value.is between the
MDL and 3 x MDL). Alternatively, the MDLs given in Appendix B,
Table B-l may be used provided that those MDLs have been achieved
and demonstrated-
15.1.1 For samples in which the quantitation ion for a given analyte
is not observed, calculate the estimated MDL by extrapolat-
ing from the analyte's quantitation ion peak area in the
unfortified or fortified sample (where the analyte is first
observed) from that day's analyses to the peak area where
peak area approaches peak height. This corresponds to the
response region where the minimum qualitative criterion
(Section 11.0) can be met. The estimated MDL is then cal-
culated as below:
Peak Area
Estimated MDL = -=—r--r— x Analyte Level (in unfortified
Peak Areass Qr fortified
sample)
where Peak Areap,_p,, is the approximate peak area where the
analyte's quantitation peak area approaches the peak height;
and Peak Area^ is the quantitation ion peak area in the
OO ' ;
daily unfortified or fortified sample.
28
-------
For example, if the analyte quantiation ion for the 100-ppt
spike level has a peak area of 5,000 area units and the
point where the peak area approaches the peak height is
approximately 1,000 units (as approximated from the cali-
bration data), the estimated MDL is calculated as shown
below:
Estimated MDL = ^ x 100 ppt = 20 ppt
Figure 5 shows the MDL determination process in flowchart
form.
15.1.2 For samples in which a response at the retention time of a
given analyte is observed, but the ion ratios differ from
these ratios observed in the daily standard by more than
30%, the estimated method detection limit is calculated as
if the peak observed were the actual analyte using the equa-
tions in Section 13.0. The values are qualified as not de-
tected, ND, and the concentration is reported in parentheses.
15.1.3 If a response for a given analyte is qualified as a trace,
TR, value (between the estimated MDL and three times the
estimated MDL), the analyst will report the estimated MDL
that was used to classify the value as a trace. This value
will come from the analysts' previous determinations of the
estimated MDL for that analyte or from Table B-l.
16.0 Reporting and Documentation
All data should be reported on a composite or single sample basis, as
required, using a data report format shown in -Figure 6. The analyst, is
required to maintain all raw data, calculations, and control charts in a
format as to allow a complete external data review.
29
-------
MDL
Determination
Manual Examination of
Analyte Ion Plot for
Unfortified Sample
Go to 100 ppt Spike Level
or Next Highest Spike
Level (If no higher spike
level, MDL cannot be
determined)
Primary and Secondary Ions
Coelure with Correct Ratio
&RT?
Analyte Not
Detected
Primary and Secondary ions
Contain at Least Two Data Points?
Calculate Ratio of*
(Peak Area where
Analyte Peak Area =
Peak Height)/(Peak
Area in Sample)
(Approximate Peak
Area where Peak
Area = Peak Height
for Analyte from
Calibration Data
Multiply Ratio Times
Amount Determined In
Unfortified or Fortified
Sample to Determine
MDL
Figure 5. MDL determination procedure.
30
-------
U.S. Environmental Protection Agency
Office of Toxic Substances
Exposure Evaluation Division
Comoosite Samnla IO»
Laboratory*
Batcn Numoar*
Analvsls Data-
Ravlaumd hy , ,
Analytes
Benzene
Eromodichlorometnane
Sromoform
Carbon tetracnicride
Chlorobenzene
Chloroform
Oibromocnloromamane
1 .2-Olcnlorobenzen9
1 ,3-Olchlorooenzene
1 ,4-Olcnlorobenzene
1.1-Oichloroethane
1 ,2-Olchtaroetfiane
1,1-Oichloroetnene
irans-l .2-Olcnioroetnene
Oichloromethane
1 ,2-Oichloroorooane
eia-l ,3-Oichloroorooene
lrans-1 ,3-OicfiloroproDene
Ethylbenzeno
Styrene
1.1.2.2-1 atracnloroethane
Tetrachloroetriene
Toluene
1,1.1-Trichloroetnane'
1,1.2-Trichloroemane
Tricnioroetnane
Trichlorofluorometnana
1.2-Xylene
1 .3-Xylene/1 .4-Xylene (3)
Data
Qualifier (1 )
.
MOL
(P9S)
National Stood Network
Analysis flegort Form
Concentration
(P9g)(2)
Sample Weignts
Individual Sample I0# Weignt (g)
Total CompositB Sample Weight (g)
Labeled Analog
of internal Std
Senzene-d,
E!ramoform-!:!C
Carbon tetracnloride-13C
Chlorobenzene-
-------
References
ACS. 1979. American Chemical Society. Safety in academic chemistry labora-
tories, 3rd ed. American Chemical Society Publication, Committee on Safety.
Keith LH, Crummett W, Deegan J Jr, Libby RA, Taylor JK, Wentler G. 1983.
Analytical Chemistry 55:2210-2218.
Long GS, Winefordner JD. 1983. Analytical Chemistry 55:712A-724A.
NIH. 1984 (March). Natl. Inst. of Health. Biosafety in microbiological and
biomedical laboratories. Washington, DC: NIH, U.S. Dept. of Health and Human
Services, Public Health Service, Center for Disease Control. HHS Pub. (CDC)
84-8395.
NIOSH. 1977 (August). Natl. Inst. Occupational Safety and Health. Carcinogens
working with carcinogens. Washington, DC: NIOSH, U.S. Dept. Health, Education,
and Welfare, Public Health Service, Center for Disease Control. DHEW Pub.
NIOSH 77-206.
OSHA. 1976 (January). Occupational Safety and Health Admin. OSHA safety
and health standards, general industry. Washington, DC: OSHA, U.S. Dept.
Labor. OSHA Pub. 2206. (29 CFR 1910).
USEPA. 1985 (January)r U. S." EnvTrdhmentaT'Pr'btection Agency. Volatile organic
compounds by isotope 'dilution GC/MS. Method 1624 Revision B. Cincinnati,
OH: USEPA.
32
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APPENDIX A
SAFETY PROCEDURES FOR HANDLING BLOOD IN THE
NATIONAL BLOOD NETWORK
A-l
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SAFETY PROCEDURES FOR HANDLING BLOOD IN THE NBN
This document is intended to be a stand-alone guideline for labora-
tory personnel working on the National Blood Network (NBN). The following
handling procedures are intended to provide maximum safety to those personnel
handling blood for the NBN. These procedures are based on procedures given
in "Biosafety in Microbiological and Biomedical Laboratories," HHS Publica-
tion No. (CDC) 84-8395,1 and in the Agent Summary Statement for the HTLV-III
virus.2 General handling procedures, receipt, log-in, storage, analysis, and
disposal of the blood samples are discussed in the following sections.
GENERAL HANDLING PROCEDURES (Biosafety Level 2)
The following practices are particularly pertinent to the handling
of blood anticipated in the NBN.
* Use of syringes, needles, and other sharp instruments should
be avoided if possible.
* Gloves should be worn by all personnel engaged in activities
that may involve skin contact with the blood.
* Generation of aerosols, splashes, and spills of blood should
be avoided.
* All laboratory'glassware, equipment, disposable materials, and
wastes must be decontaminated before washing, discarding, etc.
* If laboratory clothing becomes contaminated with blood, it
must be decontaminated before being laundered or discarded.
* Work surfaces should be decontamianted at the end of each day
or when overtly contaminated. .
RECEIPT OF BLOOD SAMPLES
Blood samples provided by the blood centers will be received by a
designated sample receiver in the specially designed sample kits. The kits/
sampling containers will consist of vial racks which will hold the blood sam-
ple vials and bagged ice which will keep the samples cold. These will be sur-
rounded by a Styrofoam box which is further surrounded by a metal shipping
container.
LOG-IN AND SAMPLE HANDLING
Upon receipt of a shipping container, the designated receiver of
the samples should complete the following activities:
A-2
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1. Place the sample container in a Class I faiosafety hood (or
average face velocity of 100 linear feet per minute).
2. Put on disposable gloves and laboratory coat.
3. Open the shipping container.
4. Check for sample leakage or breakage of the sample vials.
a. If leakage or broken vials are discovered, unbroken vials
should^be retrieved and the exterior of each vial decontaminated with a chem-
ical disinfectant. Wiping the surface of or immersing the vial racks in a
1:10 solution of household bleach for 5 min will decontaminate the surfaces
in question. The remainder of the interior of the sampling container, including
broken vials, racks, documents, etc., will also be decontaminated before being
discarded.
b. If no leakage or breakage is discovered, the receiver
should continue with sample storage.
SAMPLE STORAGE
_. After log-in, samples should be stored according to the designated
storage"condvt.To'ns"'for each sample type. Those conditions are:
1. Semivolatiles: -10°C
2. Volatiles: Seal in wide-mouth jar with activated charcoal,
then store at 4°C
3. Trace elements: -10-°C
Each sample will be stored, under the conditions given, in the
sample prep laboratory for that particular sample type. After storage of the
samples, the receiver should remove his/her disposable gloves and disinfect
and discard them according to the disposal procedures described subsequently
in this document.
ANALYSIS
1. General considerations.
* Personnel working with the blood should follow BSL 2 safety
procedures described in the general handling" procedures sec-
tion of this document.
* All glassware that has come into contact with the blood sam-
ples should be immersed in a 1:10 solution of household bleach
for 5 min before being washed or discarded.
A-3
-------
* Compositing of individual blood samples into the composite sam-
ple for analysis should be conducted in a Class A hood follow-
ing BSL 2 procedures.
2. Semivolatile analysis.
* Since the blood is pH-adjusted to very low and then very high
pHs in this method, no biological activity is expected in the
samples after pH adjustment.
3. Volatile analysis.
* During the purge step, the purge gas flow from the vent port
of the two-way six-port valve should be vented to a Class A
hood or allowed to bubble through a 3% H202 solution before
being vented into the room.
4. Trace elements analysis.
* Because of the acid digestion step in this analysis, no bio-
logical activity is expected in these samples after acid
digestion.
DISPOSAL
The procedures listed below will be followed in matters regarding
disposal.
Disposable pipettes, gloves, sample vials, etc., will be im-
mersed in a 1:10 solution of commercial bleach (e.g., Clorox®)
and placed in an autoclavable container, autoclaved, and dis-
posed of as solid waste according to MRI safety procedures.
Blood from the volatile analysis will be mixed in equal volume
with a 1:10 solution of household bleach, the solution and con-
tainer autoclaved, and then disposed of as nonflammable waste
according to MRI safety procedures.
Blood from the semivolatile analysis will be disposed of as
alkaline waste according to MRI safety procedures.
Blood from the trace elements analysis will be disposed of as
acid waste according to MRI safety procedures.
Laboratory coats contaminated with blood must be soaked in
a 1:10 solution of household bleach before being laundered
or discarded.
A-4
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Solution of household bleach (dilute 1:10) used for the decon-
tamination of glassware will be disposed of by draining into
an autoclavable container, autoclaving the container and solu-
tion, and disposing of them according to MRI safety procedures.
Organic solvents will be disposed of according to MRI safety
procedures.
REFERENCES
1. CDC-NIH. 1984. U.S. Department of Health and Human Services. Public
Health Service. Centers for Disease Control and National Institute of
Health. Biosafety in microbiological and biomedical laboratories. HHS
Publication No. (CDC) 84-8395. U.S. Government Printing Office.
2. CDC. 1986. Centers for Disease Control. Human T-lymphotropic virus '
type Ill/lymphadenopathy associated virus: agent summary statement.
Journal of the American Medical Association 256(14):1857, 1861, 1868,
1873.
A-5
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APPENDIX B
PRECISION AND ACCURACY DATA
B-l
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Table 8-1. Estimated MDL and Method PerfOi-mance (Accuracy and Precision)
From Four Level by Four Replicate Study3
Compounds
Estimated
MOL
(pg/mL)
Percent"
accuracy
Precision
RSD (%) at spike level
100 pg/mL 500 pg/mL 1000 pg/mL
Benzene 100
Sronad'ichloromethane 100
Brofflofona 100
Carbon tatrachloridn 50
Chlorobenzene 100
Chloroform . 50
01broraochloromethane 50
1,2-Oichlorobenzene 100
1,3-Oichlorobenzane 100
1,4-Oichlorobenzena 100
1,1-Qfchloroethane SO
1,2-OichloroeEhane 50
trans-1.2-0ichloroethana 100
1,2-Qichloropropane . 100
£is-l,3-0ichloropropene * 200
trans-l,3-Qichloropropene' 300
Sthylbenzene 100
Styrene 50
1,1,2,2-Tetrachloroethana 100
!,I,i-Trichloroethane 100
1,1,2-Trichtoroathane 100
Trichloroethene 100
l,2;Xylene 100
l,3-Xylene/l,4-Xylene 100
1,1-Oicnloroethene9 50'
Oichloromethana
rrichlorofluoromethane9 25
88
- 90
105
96
91
91
120
109
107
116
103
108
107
81
. 79
54
79
72
134
71
82
95
72
96
29
10
14
26
35
9
10
13
33
12
9
17
36
50
6
41e
NO8
NO
15
87
12
15
12'
9
16
13
SO
58
13
31
10
14
6
13
36
22
4
14
26
22
10
17
20
30
13
8
31
21
8
17
10
23
23
47
7
44
10
0
3
5
37
13
16
12
13
9
7
19
7
24
5
27
18
16
10
22
3
6
20
54
^Analyses performed on a Finnigan OWA GC/MS system.
fiAs .measured by the slope of the regression equation.
The upper 95% confidence limit on the RSD is approximately 2 x RSD (4 replicates at each spike level,
.four replicate nonfortified blood samples were the fourth level).
Labeled analog not available; quantitated relative to internal standard method.
-NO = not detected (not included in calculation of accuracy).
C_is_-l,3-0ichloropropane spiked at 39 pg/mL, 195 pg/mL, and 390 pg/mL. ,
trans-1.3-0ich1oropropane spiked at 61 pg/mL, 305 pg/mL, and 610 pg/mL.
3These compounds, calculated by the internal standard method, do not meet the accuracy critarian of
^50-150%. Report as qualitatively present.
High background levels of dich'loromethane precluded determinations of accuracy, and .precision from
spiked blood report as qualitatively present.
. B-2
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CO
to
Table B-2. Estimated MDL and Method Performance (Accuracy and Precision)
From Three Level by Three Replicate Study3
Compound
Bromodichloromethane .
Dibromochloromethane
Styrene
Tetrachl oroethene
Toluene
1,1,1-Trichloroethane
.Analyses performed on
Estimated
MDL
(pg/mL)
100
50
50
100
100
100
a Finnigan OWA GC/MS
. Fortification levels
Percent
accuracy
82
78
102
96
81
99
system.
Spike 1
(pg/mL)
200
200
1,000
1,500
20,000
2,500
Spike 2
(pg/mL)
500
500
2,500
3,750
50,000
•6,250
Precision0
RSD (%) at
Level 1
29
17
14
5
17
12
spike level
Level 2
33
61
10
11
42
16
• *~ L.^VA w vi i wv* §^jf fc-nv. «a i up^ ui 1*1 ic i cy i troo I ut
The upper 95% confidence limit on the RSD is approximately 2 x RSD (triplicates at each spike level,
dtriplicate nonfortified blood samples were the third level.)
Labeled analog not available; quantitated relative to internal standard method.
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TECHNICAL REPORT DATA
(Please read Inaructions on the reverse before completing)
, REPORT NO.
EPA-560/5-87-008
3. RECIPIENT'S ACCESSIONING.
4. TITLE AND SUBTITLE
Volatile Organic Compounds in Whole Blood - Determina-
tion by Heated Dynamic Headspace Purge and Trap Isotope
Dilution 6C/MS
5. REPORT DATE
July 1987
6. PERFORMING ORGANIZATION CODE |
8822-A01
7. AUTHOR(S)
Paul H. Cramer, Kathy E. Boggess, John
8. PERFORMING ORGANIZATION REPORT NO.
Hosenfeld
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
10. PROGRAM ELEMENT NO.
Work Assignment No. 22
11. CONTRACT/GRANT NO.
68-02-4252
12. SPONSORING AGENCY NAME AND ADDRESS
Field Studies Branch (TS-798), Office of Toxic: Substance
U.S. Environmental Protection Agency
401 M Street, S.W.
Washington, DC 20460 '
13. TYPE OF REPORT,AND PERIOD COVERED
13. TYPE O.F REPORT AND PERIQI
s Special (11/84-8/87)
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
The EPA Work Assignment Manager is Janet C. Remmers (202) 382-3582.
The EPA Project Officer is Joseph J. Breen (202) 382-3569.
16. ABSTRACT •• '
The method described here was developed for the determination of a selected list
of 30 organic compounds in whole human blood. The method is a heated dynamic head-
space purge and trap gas chromatographic method using mass spectrometry in the lim-
ited mass scan mode. A whole blood sample is diluted with organic free water and the
mixture fortified with isotopically labeled compounds. The mixture is subsequently
heated to 50°C while stirred and the volatile components are purged from the mixture
and collected on an adsorbent trap. The volatile compounds are thermally desorbed
from the trap onto a wide-bore fused silica capillary column. Detection of the com-
pounds is accomplished using mass spectrometry in the.limited mass scan mode where
only selected ions are scanned.
Quantitation of. the analytes is accomplished by using the specific ion responses
from standards of the analytes and their corresponding labeled analogs or internal
standards. The response of the labeled compounds are used to correct the variability
of the analytical technique through use of an isotope dilution calculation procedure
or an internal standard calculation procedure.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lOENT!F!ERS/OPEN'ENDEO TERMS
c. COSATI Fieid/GrouD
Volatile organics
Determination
Whole blood
Isotope dilution
GC/MS
Purge and trap
3. DISTRIBUTION STATEMENT
Unlimited
19. SECURITY CLASS /This Report)
Unclassified
21. NO. OF PAGES
44
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (R«». 4_77) PREVIOUS EDITION is OBSOV.ETE
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