NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

NIOSH Manual of Analytical Methods (NMAM) 5th Edition 

BACKUP DATA REPORT 

NIOSH Method No. 8007

Title: Toluene in blood

Analyte: Toluene

Author/developer: Lee C. Stone and 

James B. Perkins

Date: 4/18/2003

 

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date. 
TOLUENE IN BLOOD

NMAM 8007, ISSUE 1 

BACKUP DATA REPORT

LEE C. STONE

JAMES B. PERKINS

FINAL 

APRIL 18, 2003 

EDITED: 

MAY 30, 2012 

CONTRACT CDC-200-2001-08000

Submitted To: 

National Institute for Occupational 

Safety and Health 

Robert A. Taft Laboratories 

4676 Columbia Parkway 

Cincinnati, Ohio 45226 

Submitted By: 

ALS Environmental, formerly 

DataChem Laboratories, Inc. 

960 West LeVoy Drive 

Salt Lake City, Utah 84123-2547
TABLE OF CONTENTS 

TOLUENE in blood 

INTRODUCTION AND BACKGROUND .................................................................................1 

REAGENTS AND MATERIALS ................................................................................................2 

Preparation of Primary Stock and Internal Standard Solutions ........................................5 

SAMPLE PREPARATION AND INSTRUMENT CONDITIONS ............................................6 

Sample Preparation ...........................................................................................................6

 Instrument Conditions .......................................................................................................7

Optimization of Instrument Conditions ............................................................................8 

 Calibration.......................................................................................................................12 

 Chromatographic Conditions ..........................................................................................13 

MATRIX EFFECTS ...................................................................................................................13 

LIMIT OF DETECTION AND QUANTITATION STUDY .....................................................15 

LONG-TERM STABILITY STUDY .........................................................................................16 

PRECISION, ACCURACY, AND BIAS STUDY.....................................................................20 

SUMMARY ................................................................................................................................21 

REFERENCES ...........................................................................................................................21 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

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TOLUENE in blood: Backup Data Report 

INTRODUCTION AND BACKGROUND 

Toluene is a clear, colorless liquid with a distinctive smell. It occurs naturally in crude oil 

and in the tolu tree. It is produced in the making of coke from coal and is a by-product in the 

manufacture of styrene. 

Toluene is utilized in a wide variety of industrial processes. It is present as a solvent in 

many commercial products such as paints, thinner, adhesives, cleaning agents, pesticides, and 

explosives; and as a cleaner and degreaser of metal [1][2]. The ubiquitous use of these chemicals 

in industry generates a large potential for excessive occupational exposure. 

Toluene has for a number of years been abused for its narcotic effect on the central 

nervous system. There are over 1,400 inhalable products available on the market, which fit into 

the categories of solvents, aerosols, gases, and nitrites. The growth in inhalant users is especially 

prevalent among the youth and in the military [3]. 

Symptoms of high toluene exposure are similar to alcohol abuse. Lower concentrations 

cause irritation of mucous membranes. Higher levels non-specifically depress the central nervous 

system and are associated with headaches, drowsiness, dizziness, confusion, and even 

unconsciousness and death. The onset of the effects varies broadly from person to person and 

seems to be influenced by prior exposure. 

After toluene is taken into the body, more than 75% of it is removed within 12 hours. It 

may leave the body unchanged during respiration or in the urine [2]. Urinary excretion of 

unchanged toluene is generally less than 5% [4]. During a 5-hour inhalation exposure to toluene, 

15% to 20% of the dose absorbed is eliminated unchanged in expired air [5]. Following a 7-hour 

exposure, about 80% of the absorbed amount of toluene is excreted as metabolites. The major 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

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elimination pathway is through the urinary metabolite hippuric acid. O-cresol, which is also a 

metabolite, accounts for less than 1% of the absorbed toluene. Metabolites are not accumulative 

over the workweek, but toluene may be, depending on the level of obesity of the exposed person 

[6]. 

The Biological Exposure Indices (BEI) Committee recommends a BEI in blood of 0.05 

mg/L for toluene [7]. 

This report evaluates a method for quantifying toluene in blood samples utilizing a gas 

chromatograph equipped with a flame ionization detector and headspace automatic sampler. The 

headspace technique is a method in which the air above a sample is injected into the analytical 

system rather than injecting the actual sample or an extract of the sample. A method based on a 

headspace technique seems ideal in this situation because the volatile nature of the analyte allows 

it to easily enter the headspace upon heating of the sample. It also eliminates the need for extra 

sample extraction steps and prevents injecting relatively dirty biological matrices onto the 

instrument where contamination and wear may occur. 

NOTE: Proper safety precautions should always be taken when dealing with any chemical 

but especially when working with biological fluids such as blood. Manipulating biological 

samples poses a serious health risk because of the potential transmittance of infectious diseases 

including hepatitis and HIV. Lab coats, goggles, and gloves must be worn at all times. Work 

should be performed in an isolated hood where possible. All waste is required by law to be 

disposed of in a properly labeled, autoclavable container. 

REAGENTS AND MATERIALS 

Presented in Table 1 is the list of reagents and solvents used for this method and its 

evaluation. Isobutanol was used as the internal standard to normalize the values 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

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TABLE 1. LIST OF CHEMICALS 

Chemical Vendor CAS # Purity Lot # 

Toluene Aldrich 108-88-3 99.8% BO 08980AO

Isobutanol Fisher 78-83-1 99.8% 85540Z

Ethylene Glycol Aldrich 107-21-1 99+% JA06146HA 

Water DataChem Laboratories 7732-18-5 ASTM Type II -- 

of toluene determined in the blood samples. Ethylene glycol was used as the solvent for 

preparing the primary stock solution. The internal standard solution was made up in water. Blood 

used in the study for standards and test samples was whole blood obtained from donation centers. 

The stock blood was stored at 4 °C in the plastic storage bag that it was originally collected in 

and sodium citrate was added at donation center to control clotting. 

In the original write up of method 8002 the primary standard solution was made up in 

acetone and the secondary standards or stock standards were made up in water. From the stock 

standards were made the working or blood standards. Because of the high instability of the water 

based stock standards a more suitable solvent was sought. It was felt that the solvent needed 

would have to be soluble in both water and toluene. Acetone seemed to be ideal but when used to 

make up the secondary standards, produced numerous artifact peaks, especially at the higher 

temperature of 99 °C. Other solvents such as ethanol and methanol were tested and were also 

found to be unacceptable for the same reasons. It was found that ethylene glycol was ideal as a 

standard solvent because of its high boiling point and low vapor pressure. Ethylene glycol, when 

used as a standard solvent, produced few artifact peaks even at 99 °C. Under the right storage 

conditions standards made up in ethylene glycol are stable for over a week (Figure 1, Table 2). 

These standards need to be stored at 4 °C in airtight glass containers having non-toluene 

permeable lids and very little headspace. 
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TABLE 2. COMPARISON OF OLD AND NEW STANDARDS MADE UP IN 

ETHYLENE GLYCOL 

Standard 

ID 

Old Standards New Standards 

Concentration

(μg/mL) 

Peak Area 

% Difference 

Date 

Made Up 

mL 

Headspace* 

Date 

Made Up 

mL 

Headspace* 

PRS 10/30/2002 6 12/31/2002 0 5.76 10.5

IS1 10/30/2002 5 12/31/2002 0 0.576 13.9

IS2 10/30/2002 4 12/31/2002 0 0.0576 14.1

IS3 11/8/2002 2 12/31/2002 0 0.00576 8.5 

* As of 12/31/2002 in 20-mL vials. 

Repeated opening of the standard containers and removal of their contents ultimately results in 

the loss toluene as can be seen in Table 2 and in Figure l. 

FIGURE 1 

COMPARISON OF OLD AND NEW STANDARDS

0

500000

1000000

1500000

01234567

ug/mL Toluene

Peak Area

OLD STD'S NEW STD'S

Originally the method called for the addition of citric acid dextrose solution (ACD). To 

evaluate the usefulness of ACD in the method, differing levels of ACD was added to headspace 

vials containing 1.5 mL of blood spiked at 1.845 ug/mL toluene and 1.5 mL of internal standard 

solution. Each level was made up in triplicate and analyzed on the headspace gas chromatograph. 

From the results there was no evidence that the ACD solution benefited the method (Figure 2). A 

literature search shed little light on the use of ACD for the determination of toluene in blood. 

Some researchers add it to blood to act as a preservative [8] and others to act as an anticoagulant 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

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[9]. Perhaps for long-term storage of samples for future analysis it might be useful, but this is 

beyond the scope of this study. As a result of these findings the addition of ACD in the method, 

was discontinued. 

FIGURE 2. THE EFFECT OF ACD ADDITION SOLUTION 

TOLUENE IN BLOOD

0

5000

10000

15000

20000

02468

mL ACD

PEAK AREA

Isobutanol

Toluene

Preparation of Primary Stock and Internal Standard Solutions 

To make the primary stock solution, a 50-mL volumetric flask was filled partially with 

ethylene glycol. With a micro liter syringe, a specific, measured volume of toluene was added to 

the flask. The 50-mL volumetric flask was brought to volume with ethylene glycol, mixed, and 

the solution was transferred to a 13 X 100 mm glass culture tube and capped with a Teflon-lined 

cap. The solution was stored at 4 °C in the dark until needed. The concentration of the analyte in 

the stock solution was calculated using the volume of analyte added, its density, its purity factor, 

and the total dilution volume. For example: 

  mLmg L mL

mg L TOLUENE /346.0

50

1 998.0 8669.0 20  



 





 













   





The original method called for the use of isobutanol as an internal standard. Because no 

complaints were registered as to the use of isobutanol it was felt that there was no need to change 

to an internal standard that was more like toluene. For this study the main function of an internal 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 6 of 22 

standard has been to help correct for partial injections, which take place when the needle in the 

headspace sampler becomes restricted. This has been a rare occurrence, but when it has 

happened the drop in the isobutanol GC peak area has been a good indication of a restricted 

needle. Other than this the internal standard could have been eliminated from the method. 

The internal standard solution was prepared by diluting 25 μL of isobutanol to 1-liter 

with de-ionized water. This was done using a 1-liter volumetric flask. The original level of 

internal standard was 40 mg/L water, but as the sensitivity of the method was improved the peak 

area of isobutanol became too great and needed to be reduced. For the method evaluation the 

internal standard concentration was 20 mg/L calculated as follows: 

  Lmg L L

mg L ISOBUTANOL /20

1

1 998.0 8018.0 25  



 





 













   





This concentration of internal standard was employed because it provided good peak shape, peak 

area, and reproducibility for the internal standard. 

SAMPLE PREPARATION AND INSTRUMENT CONDITIONS 

Sample Preparation 

The samples are prepared by first adding 1.5 mL of the internal standard solution to a 20-

mL Perkin Elmer HS-40 headspace vial. Then a graduated Luer-Lok syringe with a twenty gauge 

needle is used to transfer 1.5 mL of the sample from its shipping vial to the headspace vial. The 

vial is then quickly sealed with an aluminum crimp-cap and PTFE/Butyl septa. The vials 

contents are then mixed and analyzed. If blood samples are not going to be transferred to 

headspace vials and analyzed immediately, they should be stored at 4 °C in their original 

shipping vials. If possible no headspace should be present in the sample container. Any 

headspace will result in toluene loss depending on the headspace volume [10] Samples shipped 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

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from the field should also be sent in completely filled containers where possible. To prevent 

toluene from escaping, the samples and standards should be opened to the air as little as possible. 

One of three approaches can be taken in sampling a vial. First, if the sample vial is 

equipped with a septum an aliquot of blood can be removed with a graduated syringe. Two 

twenty gauge needles can be used, one for the syringe and the other to allow air to displace the 

sample when being withdrawn. Second, using one needle, air can be injected into the vial with a 

syringe before the sample is withdrawn. Third, the lid is removed and using a macro pipette an 

aliquot of blood is removed. Glass beads can then be added to eliminate the headspace and the 

container is recapped. Once a headspace is created after an aliquot is removed a second sampling 

may result in lower values. This depends on the length of time between samplings, the volume of 

the headspace, and the amount of sample remaining in the vial. 

Instrument Conditions 

All of the samples and standards were run on the same system with the same set of 

conditions. The system consisted of a Perkin Elmer Autosystem gas chromatograph equipped 

with a FID detector and a Perkin Elmer HS-40 headspace auto sampler. The column is a fused 

silica capillary column (DB-624, 75m X 0.53mm I.D., 3.0 μm film). Figure 3 shows the 

temperature program that was utilized. The head pressure was maintained at 21 psi. The 

FIGURE 3. TEMPERATURE PROGRAM 

 220 C 

 10 /min 5min 

50 C 

2min 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

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carrier gas consisted of pre-purified helium and the FID detector was supplied with pre-purified 

hydrogen and filtered air. The injector and detector temperatures were 129 °C and 250 °C 

respectively. The injection conditions were split-less or direct on column injection for one 

minute then the splitter was turned on with a flow of 130 mL/min. Failure to use the splitter in 

this method, results in heavy carry over for both the internal standard and toluene. The headspace 

auto-sampler was set to the following conditions: 

Transfer Temp: 129 C Withdrawal: 0.80 min 

 Thermostat Time: 30 min Needle: 120 C 

 GC Cycle Time: 26 min Sample: 99 C 

 Pressurize: 4.0 min Inject: 0.4 min 

Table 3 shows the typical retention times of the analytes given these conditions. The final 

TABLE 3. ANALYTE RETENTION TIMES 

Analyte Retention Time (min)

Isobutanol 6.53 

Toluene 9.11 

temperature (220 °C) was maintained until the column appeared to be clean in order to help 

avoid any carryover into the next sample injection. To insure that there was no carry over from 

the high concentration standards to other standards or samples, headspace vials containing only 

water were inserted in the run after the high standard. A visual examination of the blank water 

runs ensured that there was no carry over of ether toluene or isobutanol. 

Optimization of Instrument Conditions 

Sample Heating Test. The conditions listed above were determined by running a battery 

of tests. In these test water was used in place of blood, because government regulations have 

made blood difficult to obtain. Headspace vials containing 1.5 mL of water and 1.5 mL of 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 9 of 22 

internal standard solution were spiked with toluene and run in sets of four. After each set was 

analyzed the conditions were changed and the next set was run. 

Heating time of 30 minutes and head pressure or 21 psi was chosen for the first test. The 

first set in this test was analyzed at 72 °C, after which the temperature was raised approximately 

10 degrees and the next set was run. This was repeated until 99 °C had been reached (Figure 4). 

The results showed that the levels of both toluene and isobutanol increased in the headspace as 

temperature increases. The effect of the higher temperatures on isobutanol eliminates the need 

for the addition of salt to the samples. For compounds that are highly water soluble, the addition 

of salt shifts their solubility from the aqueous phase to gaseous phase [11], thus improving peak 

response. The recommended head pressure listed in the operations manual for the HS-40 is 21 

psi for a sample temperature of 99 °C. This pressure was chosen because the highest temperature 

of the test was 99 °C. Because of concern for running the headspace sampler at this temperature 

the boiling points of blood and water were checked. This was done using an oil bath, hot plate, 

and thermometer. At the lab elevation of 4000 ft it was found that on the day of the test, water 

boiled at 97 °C and blood showed weak signs of boiling at 105 °C. For the headspace vials used 

in this test there were no indications of pressure problems for either blood or water at 99 °C*. 

*NOTE: For water to boil at 100 °C the atmospheric pressure would have to be 14.7 psi. The 

method uses a head pressure of 21 psi. At this pressure water would have to be heated to 111 °C 

before boiling would occur, this temperature is well above the temperature of 99 °C used in the 

method.
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FIGURE 4 

TEMPERATURE EFFECTS

0

200000

400000

600000

800000

70 80 90 99

DEGREES C

PEAK ARE

A

ISOBUTANOL

TOLUENE

Heating Time Test. Having established the operating temperature and head pressure, the 

next step was to establish the heating time needed for the samples. Spiked water samples again 

were run in sets of four varying only the time the samples were heated (Figure 5). Both toluene 

and isobutanol increase in the headspace with increased time up to 30 minutes. It was felt that the 

test did not need to go beyond 30 minutes. 

FIGURE 5 

HEATING TEST 21PSI 99DEG

0

200000

400000

600000

800000

10 20 30

MINUTES

PEAK ARE

A

ISOBUTANOL

TOLUENE

Injection and Pressurization Test. The Perkin Elmer headspace system does not use 

sampling loops. In this case the amount of sample that is transferred to the column is time and 

pressure controlled. The sample vial is first pressurized, for a set time, through the injection 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 11 of 22 

needle. Then the carrier gas is interrupted and the pressurized gas in the sample vial is allowed to

backflow out of the vial and onto the column for a set amount of time. Having determined the 

head pressure the optimum time of injection and pressurization was needed. 

The pressurization test was set up so that sets of four spiked water samples were run at 

four different times from one minute to four minutes (Figure 6). Although there was not a great 

increase in peak area from one minute to four minutes, four minutes was selected at the 

recommendation of Perkin Elmer based upon the belief that the longer time would result in 

improved precision. 

The injection test was run from 0.08 to 0.6 minutes again using spiked set of water 

samples (Figure 7). As can be seen in this injection test, time has a significant impact on peak 

response. The injection time of 0.4 minutes was chosen because times greater than this showed 

indications of column overload at higher concentrations of toluene and isobutanol. 

FIGURE 6 

PRESSURIZATION EFFECTS

0

200000

400000

600000

800000

1234

MINUTES

PEAK AREA

ISOBUTANOL

TOLUENE
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 12 of 22 

FIGURE 7 

INJECTION STUDY

0

500000

1000000

1500000

0.08 0.16 0.24 0.3 0.4 0.5 0.6

INJECTION TIME

PEAK ARE

A

ISOBUTANOL

TOLUENE

Calibration

To quantify the amount of toluene present in samples, a calibration curve should be 

constructed daily with at least five standards made up in blood, run in duplicate and covering the 

anticipated concentration range of the samples. The blood standards are prepared by adding 1.5 

mL of the internal standard solution and 1.5 mL of the stock blood to a 20-mL headspace vial 

and then spiking known amounts of the toluene/ethylene glycol working stock solution into each 

using a graduated micro-syringe. To avoid loss of toluene the spike should be made either under 

the solution or after the vial has been capped through the septum. Along with the working 

standards at least two blood blanks need to be prepared by following the same procedure only 

omitting the toluene spike. The same stock blood used for making the blood standards should be 

used for the blood blanks. 

After analyzing the samples and standards, a calibration graph is generated for toluene by 

plotting the ratio of isobutanol and the blank corrected toluene (peak area of toluene minus the 

blank divided by the peak area of the internal standard on the same chromatogram) on the y-axis 

vs. μg of toluene/mL of blood on the x-axis. A linear or quadratic model is then generated using 

this data depending on which model provides the best fit. The concentration of toluene in the 

samples is then calculated from the ratio of its toluene peak area and its internal standard peak 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 13 of 22 

area. Of the stocks of blood tested and used there has been a blank peak response in the range of 

0.005 μg/mL toluene for each. 

Chromatographic Conditions 

Figure 8 displays a typical chromatogram when the previously described conditions are 

employed. Isobutanol was chosen as the internal standard because of its availability and stability 

in water. It also elutes in a relatively interference-free region of the chromatogram. It may appear 

that the initial temperature program ramp begins too slowly and that higher initial temperatures 

and ramps would decrease the analysis time. Although this may be the case, it is not 

recommended at very low levels of toluene. A temperature program that has a steeper gradient 

may cause overlapping and interfering peaks in some samples. 

FIGURE 8. TYPICAL CHROMATOGRAM 

MATRIX EFFECTS 

When toluene standards made up in water are compared to toluene standards made up in 

blood a large bias can be seen. Compared to water, toluene in blood is approximately 40% lower 

in peak area. To see if this bias was due to a component of blood, an aliquot of whole blood was 

spun down and the two fractions were separated (plasma only and red blood cells). Each fraction 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 14 of 22 

was sampled four times and each sample was spiked and analyzed. The average results show a 

significant bias between the fractions (Table 4). The results indicate that the red blood cells may 

have an affinity for both toluene and isobutanol. 

By adding fibrous material to the headspace vials containing blood, recoveries equaling 

that of water standards can be achieved (Figure 9). When heated the blood samples and standards 

quickly gel. This gelatinous phase may act to inhibit the diffusion of toluene into the headspace. 

The addition of fibrous material, such as glass fiber filters, increases the surface area of the 

blood, thus improving diffusion to the headspace and effectively diminishing the matrix effect. 

Other fibrous materials such as Whatman filters and cotton gauze were tried but these 

contributed significant background contamination. 

TABLE 4. COMPARISON OF TOLUENE AND ISOBUTANOL RECOVERY FROM 

BLOOD AND PLASMA 

Isobutanol Toluene 

Plasma Blood Plasma Blood 

429920 370209 425723 372735 

420984 363803 440821 367175 Peak Area 433598 370128 467014 386693 

435936 361061 459521 396225 

Average 430110 366300 448270 380707 

% Difference 17.4 17.7 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

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FIGURE 9. MATRIX EFFECTS ON TOLUENE IN BLOOD AND WATER 

TOLUENE IN BLOOD AND WATER

0

200000

400000

600000

800000

1000000

1200000

02468

ug/mL Toluene

Toluene Peak Area

Water

Blood on glass

filters

Blood no filters

LIMIT OF DETECTION AND QUANTITATION STUDY 

This study was to determine the limit of detection (LOD) of the method based upon 

blood spiked with toluene. The limit of quantitation (LOQ) for toluene was then calculated as 

10/3 times the LOD. The resulting LOQ was then used to determine the target concentrations for 

all subsequent steps in the method evaluation. 

Blood standards were prepared in duplicate by spiking headspace vials containing 1.5 mL 

of blood and 1.5 mL of internal standard solution with aliquots of the stock standards (described 

in Reagents and Materials section). Table 5 lists the range of concentrations used during the 

study. The samples were then prepared and analyzed as described in the Sample Preparation and 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 16 of 22 

TABLE 5. LOD/LOQ STANDARDS 

Standard Toluene (μg/mL) 

$1 5.76

$2 2.08

$3 0.576

$4 0.208

$5 0.0576

$6 0.0208

$7 0.00576

$8 0.00208

Instrument Conditions section. The LOD and LOQ were estimated by fitting the data to a 

quadratic curve followed by applying Burkart’s Method to the data [12]. The correlation 

coefficient for toluene was 0.999 for the quadratic curve. The data provided an LOD estimate of 

0.006 μg/mL for toluene. This equates to a LOQ of 0.02 μg/mL. 

This experiment was repeated three different times on three different days to determine if 

the LOD was consistently achievable. The LODs ranged from 0.003 to 0.008 μg/mL. Although 

lower LODs were occasionally obtained, 0.006 μg/mL was chosen as the method’s LOD because 

it was consistently obtainable. 

LONG-TERM STABILITY STUDY 

This experiment is designed to assess the stability of toluene in blood under various 

conditions of storage. The study covers a period of 30 days with samples stored both at room 

temperature (24 °C) and under refrigeration (4 °C). The study was performed using whole blood 

for both samples and standards. For samples to be considered stable, they must have a loss of 

less than 10% over a period of seven days [13]. Once at the laboratory, the analysis might be 

further delayed for various reasons; therefore, the study was extended for 30 days. 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 17 of 22 

More than one attempt at running the stability study was made. It was found that blood 

could not be spiked with toluene and then transferred to storage vials without experiencing 

substantial loss. An open vial or any headspace can result in the escape of toluene from the 

sample. To resolve the problem 2-mL GC vials were filled with 1.5 mL of blood, and the vials 

were sealed with standard crimp on aluminum septum caps. Next the blood in the vials was 

spiked with toluene by injecting the blood through the septum using a syringe. To stop any loss 

of toluene through the puncture hole a dab of silicon rubber was then applied to the top of the 

septum. These vials were then stored at either 24 °C or 4 °C. At the time of analysis the 2-mL 

vials were first covered on the bottom end with a one inch square piece of aluminum foil. This 

was done, by centering the vial on the aluminum foil square. Using the thumb and forefinger the 

foil was smoothed and wrapped over the bottom and around the sides of the vial. The vials were 

then placed upside down inside a 20-mL headspace vials to which 1.5 mL of internal standard 

solution was then added. Using a center punch and small hammer the bottom of the 2-mL vials 

was then shattered and the headspace vials was quickly capped and sealed. The purpose for the 

aluminum foil was to stop the blood inside of the 2-mL vial from splashing out when the vial 

was shattered. The headspace vial was next shaken and rotated till the contents were fully mixed 

and the aluminum foil was at the bottom of the headspace vial. Because of extra surface area 

created by the addition of the 2-mL vials and aluminum foil the results would tend to be biased 

high (see section on Matrix Effects), therefore the standards were also prepared, at the time of 

the analysis, in the same manor. The spike level was set at 0.161 ug/mL (just below 10 x LOQ). 

Thirty samples and seven blanks were individually made up. On each scheduled day the number 

of samples needed plus one blank were removed from storage and analyzed. With each sample 

set, a fresh series of standards was prepared and analyzed to produce a calibration curve. Table 6 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 18 of 22 

shows the schedule for the analysis of the samples stored at room temperature (24 °C), the length 

of storage and the results for each set of samples. 

TABLE 6. LONG TERM STABILITY STUDY AT ROOM TEMPERATURE (24 °C) 

Amount Target Value Storage Storage Recovered % Recovered Sample # μg/mL Time Temperature μg/mL (Toluene) (Toluene) (Toluene) 

1 Day 1 24 C 0.161 0.158 98.1 

2 24 C 0.161 0.158 98.1 

3 24 C 0.161 0.155 96.3 

4 24 C 0.161 0.141 87.6 

5 24 C 0.161 0.156 96.9 

6 24 C 0.161 0.152 94.4 

7 Days 1 24 C 0.161 0.157 97.5 

2 24 C 0.161 0.155 96.3 

3 24 C 0.161 0.157 97.5 

4 24 C 0.161 0.151 93.8 

5 24 C 0.161 0.147 91.3 

6 24 C 0.161 0.165 102.5 

Average 0.1544 95.9 

Std. Dev. 0.006 3.8 

The samples are stable for seven days at room temperature as long as they do not clot. 

Blood used in this study was citrate treated to prevent clotting (see Reagent and Background 

section). Field samples if collected in untreated vacuum blood collection vials will clot in a 

matter of hours even if they are refrigerated. For this reason blood collection vials need to be 

treated with an anti-clotting agent. 

To determine the stability of the blood samples for longer terms of storage, up to 30 days, 

an experiment was performed as per the recommendations from the “NIOSH Guide for Air 

Sampling and Method Development and Evaluation” [13]. The 2-mL vial blood samples were 

divided into one group of six, and four groups of three. Since the data from the one-day storage 
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 19 of 22 

at room temperature was already performed in the previous experiment, it was not deemed 

necessary to repeat the experiment a second time. The eighteen samples were refrigerated at 4 

°C. The group of six samples was analyzed after seven days. The remaining four groups of three 

samples each were analyzed after 10, 14, 21, and 30 days. Results from the refrigeration study 

are presented in Table 7. 

TABLE 7. LONG TERM STABILITY STUDY AT 4 °C 

Storage 

Time Sample # Storage Temperature 

Target Value 

μg/mL 

(Toluene) 

Amount 

Recovered 

μg/mL 

(Toluene) 

% Recovered 

(Toluene) 

7 Days 1 4 C 0.161 0.165 102.5

2 4 C 0.161 0.156 96.9

3 4 C 0.161 0.154 95.7

4 4 C 0.161 0.171 106.2

5 4 C 0.161 0.162 100.6

6 4 C 0.161 0.168 104.3 

10 Days 1 4 C 0.161 0.158 98.1

2 4 C 0.161 0.162 100.6

3 4 C 0.161 0.156 96.9 

14 Days 1 4 C 0.161 0.162 100.6

2 4 C 0.161 0.153 95.0

3 4 C 0.161 0.148 91.9 

21 Days 1 4 C 0.161 0.158 98.1

2 4 C 0.161 0.158 98.1

3 4 C 0.161 0.158 98.1 

30 Days 1 4 C 0.161 0.162 100.6

2 4 C 0.161 0.159 92.5

3 4 C 0.161 0.157 97.5

Average 0.159 98.6

Std. Dev. 0.005 3.6
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 20 of 22 

The results from the long-term stability study show that toluene is stable in blood for at 

least 30 days as long as they are kept at 4 °C with no headspace in the sample container and the 

lid is well sealed. 

PRECISION, ACCURACY, AND BIAS STUDY 

A study was performed to determine the precision, accuracy, and bias of the method 

using whole blood. The bias for a method must be less than or equal to 10%. The coefficient of 

variation (CV) must be less than or equal to 0.1. When these conditions are met the method is 

deemed unbiased and accurate with 95% probability to within ±25% of the actual concentrations 

[13]. 

During the study, six concentrations ranging from 1 x LOQ (0.02 μg/mL) to 300 x LOQ 

(6.0 μg/mL) were studied. Whole blood (1.5 mL) was used for each sample. Six samples at each 

level (a total of 36 samples) were spiked with toluene and isobutanol solutions (described in 

Reagents and Materials section) to produce the desired concentrations. The samples were then 

prepared and analyzed according to the method. Table 8 displays the average recoveries of the 

analytes over the ranges studied. 

TABLE 8. AVERAGE RECOVERIES OVER VARIOUS LEVELS 

Level (xLOQ) Rough Target 

(g/mL) 

Actual Spike

(g/mL) 

Average %

Recovered CV 

Toluene Toluene Toluene

1 0.02 0.02076 105.9 0.044

3 0.06 0.0576 97.0 0.037

10 0.2 0.2076 98.7 0.008

30 0.6 0.576 100.9 0.025

100 2 2.076 100.1 0.014

300 6 5.76 100.6 0.010
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 21 of 22 

The data were processed to determine the coefficient of variation (CV) at each 

concentration level and whether these levels could be pooled using Barlett’s test of homogeneity 

[13]. The pooled coefficients of variation of those that passed the 95% test were then used for 

calculating the overall precision and accuracy of the method as presented in Table 7. The 1 x 

LOQ level was not included in the calculations presented in Table 9. 

Bias Precision

Analyte Range (μg/mL) Accuracy (%) Average Range Overall (ŜrT) 

Toluene 0.0208 to 5.76 <5 0.0006 -0.013 to 0.0085 0.0157 

TABLE 9. PRECISION, BIAS, AND ACCURACY 

SUMMARY 

All data derived during the method development met all NIOSH criteria for precision, 

bias, and accuracy in all studies performed [13]. The method proved to be rugged and adaptable 

to human blood samples. 

REFERENCES 

[1] Toluene. (n.d.). Retrieved January 15, 2003, from

http://wey238ab.ch.iup.edu/olccii/student/read.html

[2] Toluene. (n.d.). Retrieved January 14, 2003, from http://www.ecousa.net/toxics/toluene.shtml

[3] Sheryl H. Peyton, MS, L. Dawn Herring and Glenn Harrison (1998, Fall Meeting)

Toluene Abuse in The Nineties. Southwestern Association of Toxicologists. Retrieved

January 14, 2003, from Austin Meeting News Web Site: http://www.sattox.org/austin.html\

[4] Ghittori, S.; Imbriani, M,; Pezzagno, G.; et al.: The Urinary Concentration of Solvents as

a Biological Indicator of Exposure: Proposal for the Biological Equivalent Exposure

Limit for Nine Solvents. Am. Ind. Hyg. Assoc. J. 48:786-790 (1987).

[5] Sato, A.; Nakajima, T.; Fujiwara, Y.; Hirosawa, K.: Pharmacokinetics of Benzene and

Toluene.Int. Arch. Arbietsmed. 33:169-182 (1974).
TOLUENE in blood: Backup Data Report Last updated: 05/30/2012 

Page 22 of 22 

[6] Cohr, K.J.; Stockholm, J.:Toluene: A Toxicological Review. Scand.J. Work Environ.

Health 5:71-90 (1979).

[7] BEI® (Biological Exposure Indices) based on 2000 Recommendations of the American

Conference of Governmental Industrial Hygienists (ACGIH).

[8] Wujanz G, Maede D (1993) Research Projects: Medical Animal Hospital. Retrieved

September 9, 2002, from Research projects: Medical Animal Hospital Web Site: http://

216.239.35.120/translate_c?hl=en&s1=de&u=http://www.unileipzig.de/forsh94/250

[9] Non-Radioactivc Reagents and Diluents. (n.d.). Retrieved January 15, 2003, from

http://www.ukrg.org.uk/rphandbook/acd.htm

[10] Gill R, Hatchett SE, Osselton MD (1988) Sample Handling and Storage for the

Quantitative Analysis of Volatile Compounds in Blood: The Determination of Toluene

by Headspace Gas Chromatography. Analytical Toxicology. J. 12: 141-146

[11] Restek Corp. (2000) A Technical Guide for Static Headspace Analysis Using GC Lit.

Cat. # 59895A

[12] Burkart JA (1986) General Procedures for Limit of Detection Calculations in Industrial

Hygiene Chemistry Laboratory. Appl Ind Hyg 1(3): 153-155.

[13] Kennedy ER, Fishbach, TJ, Song R, Eller PM, Shulman, SA (1995) Guidelines for Air

and Analytical Method Development and Evaluation. Cincinnati, OH: National Institute

for Occupational Safety and Health, DHHS (NIOSH) Publication No. 95-117.

Written By: Lee C. Stone and James B. Perkins, DataChem Laboratories, Inc, Salt Lake City, 

Utah under NIOSH CDC Contract 200-2001-08000. 

Edited By: John M. Reynolds and Roxanne W. Olson, ALS Environmental, formerly DataChem 

Laboratories, Inc., Salt Lake City Utah.