NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

NIOSH Manual of Analytical Methods (NMAM) 5th Edition 

BACKUP DATA REPORT

NIOSH Method No. 8318

Title: 2,5-Hexanedione in urine

Analyte: 2,5 Hexanedione

 Author/developer: Corey Downs, James Perkins

 Date: November 9, 2015

Disclaimer: Mention of any company or product does not constitute endorsement by the National 

Institute for Occupational Safety and Health, Centers for Disease Control and Prevention. In addition, 

citations to websites external to NIOSH do not constitute NIOSH endorsement of the sponsoring 

organizations or their programs or products. Furthermore, NIOSH is not responsible for the content of 

these websites. All web addresses referenced in this document were accessible as of the publication 

date. 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

2,5-HEXANEDIONE in urine: Back-up Data Report

INTRODUCTION AND BACKGROUND

n-Hexane is not a normal constituent of the environment although it can be present in 

gasoline, paint, glue, and ink. In the workplace, n-hexane is generally present as a vapor 

where its major form of absorption is via inhalation—although a significant dermal 

absorption of the liquid is possible as well. Prolonged n-hexane exposure can lead to neural 

diseases such as peripheral neuropathy [1]. Consequently, monitoring exposure to n-hexane, 

through urinalysis, continues to be an important part of exposure assessment. A measurement 

of n-hexane exposure is possible by monitoring the urinary levels of its major metabolite, 

2,5-hexanedione [2]. In fact, the neurotoxicity of n-hexane seems to stem from 2,5-

hexanedione and its ability to interact with ε-amino groups of lysine in proteins [3]. The 

production of 2,5-hexanedione is not completely specific to n-hexane exposure; 2,5-

hexanedione is also a metabolite of methyl n-butyl ketone. It also has been shown that nhexane metabolism is inhibited by coexposure to toluene and methyl ethyl ketone [4].

The method for analyzing 2,5-hexanedione evaluated in this report is based on an acid 

hydrolysis and liquid extraction method of Iwata et al. [4], and Fedtke and Bolt [5], as 

described by Kawai et al. [6]. Acid hydrolysis can cause more than a tenfold increase in the 

amount of 2,5-hexanedione detected. n-Hexane is metabolized into several intermediates 

besides 2,5-hexanedione. Most of these intermediates undergo further transformation and are 

excreted as glucuronide conjugates. 2,5-Hexanedione, however, is not conjugated; it is 

referred to as the “free” amount of 2,5-hexanedione. Acid hydrolysis releases n-hexane 

metabolic intermediates from their conjugation but also converts some of the now freed 

intermediates (most notably 4,5-dihydroxy-2-hexanone) into 2,5-hexanedione [7]. The 

conversion of n-hexane intermediates into 2,5-hexanedione, via acid hydrolysis, significantly 

increases the amount of 2,5-hexanedione detected. The “free” amount of 2,5-hexanedione 

plus the extra amount gained from conversion of intermediates is deemed the “total” amount 

of 2,5-hexanedione. This method measures the “total” amount of 2,5-hexanedione. 

The reactions for freeing n-hexane intermediates and converting them to 2,5-

hexanedione are pH dependent. A pH of ≤ 1 is critical for driving these reactions to 

completion and thus producing consistent and reproducible results when trying to quantify 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

the “total” amount of 2,5-hexanedione present in a sample [4]. Acid hydrolysis can also 

cause an elevated 2,5-hexanedione background level in pooled urine blanks. 

Work by Kawai et al. [6] suggests that acid hydrolysis may not be necessary and that 

more accurate results are obtained when only “free” 2,5-hexanedione is measured by 

skipping the hydrolysis step. They found a much closer correlation between n-hexane 

exposure by inhalation (as measured by personal samplers) and 2,5-hexanedione in urine 

when the acid hydrolysis step was omitted rather than included (correlation coefficient of 

0.867 vs. 0.344). Furthermore, the background level of pooled urine blanks was zero in the 

absence of acid hydrolysis. Because of these findings, guidelines published by various 

organizations have taken different approaches. 

The Biological Exposure Indices (BEI) Committee of the American Conference of 

Governmental Industrial Hygienists (ACGIH) recommends the Kawai approach and sets the 

BEI at 0.4 mg/L of “free” 2,5-hexanedione measured without acid hydrolysis [8]. The BEI 

change from “total” to “free” took place in 2002-2003, shortly after the development of this 

method. European organizations have continued to use an acid hydrolysis approach but with 

some modifications. The German recommendation for the biological tolerance value (BAT) 

set forth by the Deutsche Forschunggemeinschaft (DFG) is to perform an acid hydrolysis and 

then add the levels of 2,5-hexanedione and 4,5-dihydroxy-2-hexanone. This combined 

concentration should not exceed 3.5 mg/L [9]. The Swiss (SUVA – Swiss National Accident 

Insurance Fund) take a similar approach by adding the amounts of 2,5-hexanedione and 4,5-

dihydroxy-2-hexanone after hydrolysis, but sets their recommended level at 5 mg/L [10]. 

Recently the Japan Society for Occupational Health published its recommendation and 

included values for 2,5-hexanedione both with (3 mg/g creatinine) and without (0.3 mg/g 

creatinine) hydrolysis [11]. 

Work by Tondel et al. [12] has recently looked at 2,5-hexanedione levels in the 

general population. Their studies of 227 persons nonoccupationally exposed to n-hexane 

showed that the mean urinary concentration for women was 0.36 mg/L and for men was 0.45 

mg/L. More studies need to be performed to determine if this is environmental exposure or 

endogenous production, but background levels should be expected and there are statistical 

differences noted by gender and by age. 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

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

chemical but especially when working with biological fluids such as urine. 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 (powder-free 

latex or nitrile) 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. 2-Methyl-3-heptanone was selected for use as an internal standard to normalize

TABLE 1. LIST OF CHEMICALS

Chemical Vendor CAS # Purity Lot #

2,5-Hexanedione Aldrich 110-13-4 98% TI 016622 LI

2-Methyl-3-heptanone Aldrich 13019-20-0 99% MO 08128 LO

Methanol Burdick & Jackson 67-56-1 HPLC BY967

Dichloromethane Burdick & Jackson 75-09-2 HPLC CD197

HCl Conc. (36-38%) JT Baker 7647-01-0 Trace Metal H39036

Uri-Sub (Synthetic Urine) CST Technologies, Inc. US080901Z

values of 2,5-hexanedione determined in the urine samples. Hydrochloric acid was utilized 

during the acid hydrolysis and methanol was used while preparing the primary stock 

solutions. Both synthetic and pooled urine were used during the study. Many methods 

developed for determination of urinary metabolites utilize water as the base matrix due to 

possible interferences during method development. DCL decided to begin experiments using 

synthetic urine to more closely study the possible matrix effects of urine and to minimize 

biological exposure during the early part of the study. Actual urine used in the study was 

collected from volunteer employees at DataChem Laboratories in Salt Lake City. The urine 

was collected and pooled as needed. Once pooled, it was stored at 4 °C in 1-L polyethylene 

screw-top bottles. The stock synthetic urine was always stored at room temperature. 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

Preparation of Primary Stock and Extraction Solutions 

To make the primary stock solutions, 10-mL volumetric flasks were tared on an 

analytical balance. With a syringe, an appropriate amount of 2,5-hexanedione (25-250 μL) 

was added to each flask while noting the increase in mass. The contents of the 10-mL 

volumetric flasks were brought to volume with methanol, mixed, and transferred to a 13 x 

100 mm glass culture tube with a Teflon-lined cap. The solution was stored at 4 °C in the 

dark until needed. This produced stock solutions containing a range of 2-20 mg/mL of 2,5-

hexanedione. Depending on the desired range needed for the study, dilutions were made from 

the appropriate primary stock solution. 

The extraction solution was made by adding 5 mg of 2-methyl-3-heptanone into 

enough dichloromethane to make a total volume of 1 L. This concentration of internal 

standard in extraction solution was employed because it provided good peak shape, peak 

area, and reproducibility for the internal standard. 

SAMPLE PREPARATION AND INSTRUMENT CONDITIONS

Sample Preparation

For each study, the samples and standards were always prepared in the same manner. 

Exactly 5.0 mL of urine, synthetic or pooled, was transferred into a 13 x 100 mm glass 

culture tube. Utilizing a magnetic stirrer and stir bar, the pH of the sample was adjusted to 

between 0.5 and 1.0 by adding HCl. As noted in the introduction, precise control of the pH is 

critical for consistent results. The tube was then capped and heated in a water bath for 30 

minutes at 100 °C. After cooling, 2 mL of the dichloromethane extraction solution, 

containing the internal standard (5 μg/mL), was added to the tube and the sample was shaken 

vigorously for about one minute. After the phases separated, the samples were spun lightly in 

a centrifuge to aid the separation. A portion of the dichloromethane extraction solution 

(bottom layer) was transferred to a 2-mL GC autosampler vial for analysis.

Instrument Conditions

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

conditions. The system consisted of an HP5890 Series II gas chromatograph equipped with a 

flame ionization detector and an autosampler. The column was a polyethylene glycol fused 

silica capillary column (DB-WAX, 30 m x 0.32 mm I.D., 0.50 μm film). Figure 1 shows the 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

temperature program that was utilized. For dirtier samples, the final hold time was extended 

to bake-out the column. The head pressure was maintained at 15 psi. The carrier gas 

consisted of pre-purified helium and the FID was supplied with pre-purified hydrogen and 

filtered air. The injector and detector temperatures were 200 °C and 250 °C, respectively. A 

3-μL aliquot of the dichloromethane extract was injected for each sample. The injection 

conditions were splitless and the purge was delayed for 0.7 minutes. Table 2 shows the 

retention times of the analytes given these conditions. The final temperature (220 °C) was 

maintained until the column appeared to be clean in order to avoid any carryover into the 

next sample injection.

FIGURE 1. TEMPERATURE PROGRAM

TABLE 2. ANALYTE RETENTION TIMES

Analyte Retention Time (min)

2-Methyl-3-heptanone 4.73

2,5-Hexanedione 10.55

Calibration 

 To quantify the 2,5-hexanedione present in samples, a calibration curve was 

constructed daily with at least six working standards covering the anticipated concentration 

range of the samples. The working standards were prepared by diluting a known amount of 

2,5-hexanedione stock solution into enough pooled urine to make a total of 5.0 mL for each 

standard. Along with the working standards, at least one pooled urine blank was prepared by 

transferring 5.0 mL of pooled urine (the same pooled urine used for creating the working 

standards) into a culture tube. The 5 mL aliquot of each working standard and pooled urine 

blank was then processed using the same procedure as described previously. 

5°/min

45°C

(2 min)

220°C

(5 min)

60°C

10°/ min 140°

50°/ min
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

After analyzing the samples, a calibration graph was generated by plotting, for each 

working standard, the normalized analyte response (peak area of analyte divided by the peak 

area of the internal standard on the same chromatogram) on the y-axis vs. mg of analyte/L of 

urine on the x-axis. A linear or quadratic model was utilized in processing the working 

standard data depending on which model provided a better fit to the data. Because acid 

hydrolysis would create detectable amounts of 2,5-hexanedione in pooled urine blanks, 

before plotting the calibration graph it was often necessary to subtract the normalized analyte 

response of the pooled urine blank from the normalized analyte response of each working 

standard. The normalized analyte response was then calculated for each sample and the 

corresponding 2,5-hexanedione concentration was read from the x-axis of the calibration 

curve (mg/L of urine). If desired, the samples can be further normalized by using their 

creatinine concentrations.

Chromatogram Comparison/Analysis 

Figures 2 and 3 display typical chromatograms when the previously described 

conditions are employed with pooled and synthetic urine, respectively. Although, as the 

figures show, the final analyte elutes after 10 to 11 minutes, an additional 10 to 15 minutes at 

a high temperature is required to purge the remaining compounds and prevent buildup in the 

column. 2-Methyl-3-heptanone was chosen as the internal standard because of its availability 

and because its retention time positioned it in a relatively interference-free portion of the 

chromatogram of the urine sample. 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. Field samples tended to be quite 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

FIGURE 2. TYPICAL CHROMATOGRAM (POOLED URINE)

variable. A more aggressive temperature program caused overlapping and interfering peaks 

in some samples. As expected, the synthetic urine chromatogram contains fewer peaks and 

the baseline is more stable. Using synthetic urine for making standards allowed much lower 

levels of the analyte to be quantified as addressed in the studies that follow. 

FIGURE 3. TYPICAL CHROMATOGRAM (SYNTHETIC URINE) 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

Synthetic urine appears to be a viable alternative to pooled urine when preparing standards 

and quality control samples. Some advantages of using synthetic urine are,

• being less of a health hazard because it is not a biological sample

• allowing for a 40X decrease in the LOD

• eliminating the need for blank subtractions 

• producing comparable percent recoveries. 

Using pooled urine, however, is advantageous because it most closely resembles the matrix 

of the field samples and may produce the most accurate results. Ultimately, it may not matter 

what type of urine is used for standards. The method calls for a comparison of a subject’s 

2,5-hexandione levels before and after exposure. The method is interested in detecting a 

relative increase between the two samples. Because the increase is relative, as long as the 

same type of urine is used as standards for both the before and after samples, the relative 

result should be the same no matter what type of standards are employed. 

LIMIT OF DETECTION AND QUANTITATION STUDY

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

both synthetic urine and pooled control urine spiked with 2,5-hexanedione. The limit of 

quantitation (LOQ) for both 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. 

Synthetic Urine Study

A range of standards for analysis was prepared in duplicate by diluting the primary 

stock solution (described in Reagents and Materials section) and spiking the appropriate 

volume into 5 mL of synthetic urine. Table 3 lists the range of concentrations used during the 

study. The samples were then prepared and analyzed as described in Sample Preparation and 

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 [13]. The correlation 

coefficient was 0.997 for the quadratic curve. The data provided an LOD estimate of 0.005 

μg/mL. This equates to a LOQ of 0.02 μg/mL.
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

TABLE 3. SYNTHETIC URINE STANDARDS

Standard [2,5-HD] μg/mL

7 0.1183

8 0.0475

9 0.0238

10 0.0190

11 0.0143

12 0.0095

13 0.0048

Pooled Urine Study

For the pooled control urine, a range of solutions for analysis was prepared in 

duplicate by diluting the primary stock solution (described in Reagents and Materials section) 

and spiking the appropriate volume into 5 mL of pooled control urine. The samples were then 

prepared and analyzed as described in Sample Preparation and Instrument Conditions 

section. Table 4 shows the range of concentrations used during the study. As expected, 

because of the acid hydrolysis, the blank contained a significant response. It was 

questionable as to whether standard 8 produced a signal larger than the average blank 

response. The LOD and LOQ were estimated by fitting the data to a quadratic curve followed 

by applying Burkart’s Method to the data [13]. The correlation coefficient was 0.999 for the 

quadratic curve. The data was analyzed both with and without including standard 8. The 

analysis was also performed both with and without blank subtractions. Table 5 contains the 

results using these various scenarios. The study was repeated several days later with similar 

results. From the data, it was determined that the LOD for this method, utilizing the 

conditions specified in Sample Preparation and Instrument Conditions section, is 0.2 μg/mL. 

The value for the LOQ is 0.7 μg/mL.

TABLE 4. STANDARD CONCENTRATIONS FOR SPIKED POOLED URINE

Standard [2,5-HD] μg/mL

1 46.6

2 11.8

3 2.37

4 1.19

5 0.466

6 0.118

7 0.047

8 0.024
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

TABLE 5. LOD/LOQ RESULTS

STD 8 

Included

Blank 

Subtraction

LOD 

(μg/mL)

LOQ 

(μg/mL)

Yes Yes 0.2 0.6

Yes No 0.1 0.5

No Yes 0.2 0.8

No No 0.2 0.6

The LOD is 40 times lower for synthetic urine than for pooled urine. The increase in 

sensitivity can be attributed to the lack of 2,5-hexanedione in the synthetic urine blank. Acid 

hydrolysis causes a blank sample of pooled urine to produce a 2,5-hexanedione peak of 

substantial area. That area, however, can vary as much as 20% between replicate blanks. 

When small concentrations of 2,5-hexanedione are present, it is difficult to determine 

whether the increase in area is due to the analyte or to the variation in the blank response. 

Because a blank sample of synthetic urine, even after acid hydrolysis, does not show a 2,5-

hexanedione response, even minute signal increases can be attributed to analyte detection and 

a lower LOD is possible. With the success of this study and the data obtained from it, it was 

then possible to perform the spiked pooled urine experiments.

LONG-TERM STABILITY STUDY

This experiment was designed to assess the stability of 2,5-hexanedione in urine 

under various conditions of storage. The study covered a period of 7 days for samples stored

at room temperature (24 °C) and 32 days for samples stored refrigerated (4 °C). The study 

was performed using pooled urine 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 [9]. 

Once at the laboratory, the analysis might be further delayed for various reasons; therefore, 

the study was extended for 32 days. 

Samples were prepared by spiking 15 μL of a 2,5-hexanedione primary stock solution 

(see Reagents and Materials section) into 5 mL of pooled control urine. The stock solution 

was prepared so that the final concentration of the analyte in each sample was 7.11 μg/mL 

(10 x LOQ). A total of 30 samples and 8 blanks were prepared. On each day, the designated 

samples plus one blank were analyzed as described in Sample Preparation and Instrument 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

Conditions section. With each sample set, a fresh series of standards were prepared and 

analyzed to produce a calibration curve. Table 6 shows the schedule for the analysis of the 

samples stored at room temperature (24 °C), the length of storage, the results for each set of 

samples, and their recoveries. All of the recoveries were nearly 100%. One sample on Day 7 

had a slightly lower recovery (95.5%) but, as noted in the table, HCl was not added to this 

sample as specified by the method. Given the closeness of this number and the fact that the 

acid addition should have little effect on recovery of a spiked sample, the value is included in 

the values calculated at the bottom of the table. Although it appears that the samples were 

stable for at least seven days at room temperature, it is recommended that the samples always 

be refrigerated to minimize the amount of bacterial growth and possible analyte degradation. 

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

Storage Time Sample #

Storage 

Temperature

Target 

Value

Amount 

Recovered

% 

Recovered Comments

1 Day 1 24 °C 7.108 7.098 99.9

1 Day 2 24 °C 7.108 6.936 97.6

1 Day 3 24 °C 7.108 7.550 106.2

1 Day 4 24 °C 7.108 7.163 100.8

1 Day 5 24 °C 7.108 7.120 100.2

1 Day 6 24 °C 7.108 7.176 101.0

7 Days 1 24 °C 7.108 6.960 97.9

7 Days 2 24 °C 7.108 7.047 99.1

7 Days 3 24 °C 7.108 7.018 98.7

7 Days 4 24 °C 7.108 7.304 102.8

7 Days 5 24 °C 7.108 6.785 95.5 No acid added

7 Days 6 24 °C 7.108 7.016 98.7

Mean 7.108 7.098 99.9

Std. Deviation 0 0.195 2.74
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

To determine the stability of the urine 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” [9]. Five mL aliquots of pooled urine 

were divided into one group of six and four groups of three. Since the data from the one-day 

storage 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. Results from the refrigerated study are presented in Table 7. The group 

of 6 was analyzed after 7 days. The remaining four groups of 3 samples each were analyzed 

after 10, 14, 21, and 32 days. Table 7 clearly shows that the analyte is stable for at least 32 

days in urine when stored at 4 °C. Although no further studies were conducted, the analyte is 

most likely stable for much longer than 32 days. 

TABLE 7. LONG TERM STABILITY STUDY AT 4 °C 

Storage Time Sample #

Storage 

Temperature Target Value

Amount 

Recovered % Recovered

7 Days 1 4 °C 7.108 7.077 99.6

7 Days 2 4 °C 7.108 7.052 99.2

7 Days 3 4 °C 7.108 7.037 99.0

7 Days 4 4 °C 7.108 7.260 102.1

7 Days 5 4 °C 7.108 7.043 99.1

7 Days 6 4 °C 7.108 7.400 104.1

10 Days 1 4 °C 7.108 7.007 98.6

10 Days 2 4 °C 7.108 6.955 97.8

10 Days 3 4 °C 7.108 7.222 101.6

14 Days 1 4 °C 7.108 6.976 98.1

14 Days 2 4 °C 7.108 7.003 98.5

14 Days 3 4 °C 7.108 7.178 101.0

21 Days 1 4 °C 7.108 7.142 100.5

21 Days 2 4 °C 7.108 7.238 101.8

21 Days 3 4 °C 7.108 7.218 101.5

32 Days 1 4 °C 7.108 7.455 104.9

32 Days 2 4 °C 7.108 7.252 102.0

32 Days 3 4 °C 7.108 7.097 99.9

Average 7.108 7.145 100.5

Std. Deviation 0 0.143 2.0
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

PRECISION, ACCURACY, AND BIAS STUDY

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

using both pooled and synthetic urine. 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 [14].

Synthetic Urine Study

For the synthetic urine study, five concentrations ranging from 1 x LOQ (0.02 

μg/mL) to 100 x LOQ (2.0 μg/mL) were studied. Five milliliters of synthetic urine were used 

for each sample. Seven samples at each level (a total of 35 samples) were spiked with a 2,5-

hexanedione solution (described in Reagents and Materials section) to produce the desired 

concentrations. A sample blank was also included at each level. The data were also analyzed 

in a similar manner. All concentration levels were included in the calculations. Table 8 

displays the average recoveries of the analyte over the ranges studied.

TABLE 8. AVERAGE RECOVERIES FOR SYNTHETIC URINE

Level

Rough 

Target

Actual Spike 

(μg/mL)

Average % 

Recovered CV

1 X LOQ 0.02 0.020 97.9 0.0140

3 X LOQ 0.06 0.062 96.4 0.0314

10 X LOQ 0.20 0.198 94.4 0.0201

30 X LOQ 0.60 0.602 95.1 0.0091

100 X LOQ 2.00 1.995 90.8 0.0128

Table 9 shows the final results after using the pooled coefficients of variation to 

calculate the overall precision and accuracy.

TABLE 9. PRECISION, BIAS, AND ACCURACY FOR SYNTHETIC URINE

Range (μg/mL) 0.02 – 2.0

Accuracy (%) 8.2

Bias

Average -0.0507

Range -0.0916 to 0.0209

Precision

Overall (ŜrT) 0.0192
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

Pooled Urine Study

For the pooled urine, five concentrations ranging from 3 x LOQ (2.1 μg/mL) to 300 x 

LOQ (210 μg/mL) were studied. A 1 x LOQ level was also included in the study to monitor 

baseline noise, but its data was not included in the final calculations. Five milliliters of 

pooled control urine were used for each sample. Seven samples at each level (a total of 42 

samples) were spiked with a 2,5-hexanedione solution (described in Reagents and Materials 

section) to produce the desired concentrations. A sample blank was also included at each 

level. The samples were then prepared and analyzed according to the protocol listed in 

Sample Preparation and Instrument Conditions section. Table 10 shows the average 

recoveries of 2,5-hexanedione over the ranges studied. The data were processed to determine 

the coefficient of variation at each concentration level and whether these levels could be 

pooled using Barlett’s test of homogeneity [14]. The 1 x LOQ level could not be pooled. 

TABLE 10. AVERAGE RECOVERIES FOR POOLED URINE

Level

Rough Target 

(μg/mL)

Actual Spike 

(μg/mL)

Average % 

Recovered CV

1 x LOQ 0.7 0.692 107.9 0.0589

3 x LOQ 2.1 2.14 97.4 0.0230

10 x LOQ 7 7.11 102 0.0187

30 x LOQ 21 21.2 102.6 0.0128

100 x LOQ 70 71.1 103.5 0.0305

300 x LOQ 210 212 101.8 0.0282

The pooled coefficients of variation were then used for calculating the overall 

precision and accuracy of the method presented in Table 11. 

TABLE 11. PRECISION, BIAS, AND ACCURACY FOR POOLED URINE

Range (μg/mL) 0.7 – 212

Accuracy (%) 5.6

Bias

Average -0.0189

Range -0.1924 to 0.0345

Precision

Overall (ŜrT) 0.0235
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

SUMMARY

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

bias, and accuracy in all studies performed [14]. The method proved to be rugged and 

adaptable to both synthetic urine and human urine samples. The method was successfully 

used in a Health Hazard Evaluation field study [15].
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

REFERENCES

[1] WHO [1991]. World Health Organization: n-Hexane. Environmental Health Criteria 

122. WHO, Geneva. 

[2] Perbellini L, Brugnone F, Faggionato G [1981]. Urinary excretion of the metabolites of 

n-hexane and its isomers during occupational exposure. Br J Ind Med 38:20-26. 

[3] DeCaprio AP, Olajos EJ, Weber P [1982]. Covalent binding of a neurotoxic n-hexane 

metabolite: conversion of primary amines to substituted pyrrole adducts by 2,5-

hexanedione. Toxicol Appl Pharamacol 65:440-450. 

[4] Iwata M, Takeuchi Y, Hisanaga N, Ono Y [1983]. A study on biological monitoring of 

n-hexane exposure. Int Arch Occup Environ Health 51:253-260. 

[5] Fedtke N, Bolt HM [1987]. The relevance of 4,5-dihydroxy-2-hexanone in the 

excretion kinetics of n-hexane metabolites in rat and man. Arch Toxicol 61:131-137. 

[6] Kawai T, Mizunuma K, Yasugi T, Uchida Y, Ikeda M [1990]. The method of choice 

for the determination of 2,5-hexanedione as an indicator of occupational exposure to nhexane. Int Arch Occup Environ Health 62:403-408. 

[7] Fedtke N, Bolt HM [1987]. 4,5-Dihydroxy-2-hexanone: a new metabolite of n-hexane 

and of 2,5-hexanedione in rat urine. Biomed Environ Mass Spectrom 14:563-572. 

[8] ACGIH [2013]. TLVs® and BEIs® based on the documentation of the Threshold Limit 

Values for chemical substances and physical agents and Biological Exposure Indices. 

Cincinnati, Ohio: American Conference of Governmental Industrial Hygienists. p. 111. 

[9] Commission for the Investigation of Health Hazards of Chemical Compounds in the 

Work Area [2011]. List of MAK and BAT values 2011; maximum concentrations and 

biological tolerance values at the workplace. Report no. 47. Weinheim, DFG: Deutsche 

Forschunggemeinschaft, Wiley-VCH Verlag GmbH & Co., p. 219. 

[10] SUVA (Swiss National Accident Insurance Fund) [2013]. Protection de la santé au 

poste de travail. Valeurs limites d’exposition aux postes de travail. 3rd Ed. p. 130. 

www.suva.ch/waswo/1903.f

[11] JSOH [2016]. Recommendation of occupational exposure limits (2016-2017). The 

Japan Society for Occupational Safety and Health. J Occup Health 58(5):489-518. 

[12] Tondel M, Murgia N, Persson B, Lindh J, Vrethem M, Fredrikson M [2011]. 2,5-

Hexanedione in the general population: environmental exposure or endogenous 

production? Epidemiology 22(1):S34-S35. 

[13] Burkart JA [1986]. General procedures for limit of detection calculations in industrial 

hygiene chemistry laboratory. Appl Ind Hyg 1(3):153-155. 

[14] NIOSH [1995]. Guidelines for air and analytical method development and evaluation. 

By Kennedy ER, Fischbach TJ, Song R, Eller PM, Shulman, SA. Cincinnati, OH: U.S. 

Department of Health and Human Services, Centers for Disease Control and 

Prevention, National Institute for Occupational Safety and Health, DHHS (NIOSH) 

Publication No. 95-117. 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

[15] NIOSH [2006]. Hazard evaluation and technical assistance report: Engineered Fabrics 

Corporation, Rockmart, GA. By Tapp L, Mattorano D, Mueller C. Cincinnati, OH: U.S. 

Department of Health and Human Services, Centers for Disease Control and 

Prevention, National Institute for Occupational Safety and Health, NIOSH HETA 

Report No. 2000-0374-2998 [www.cdc.gov/niosh/hhe/reports/pdfs/2000-0374-

2998.pdf].
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

APPENDIX

Review of User Check for NMAM Method 8318 (2,5-Hexanedione in urine) 

User check samples were prepared by a BHAB researcher (Dr. Clayton B’Hymer) to be 

analyzed by ALS Environmental using draft NMAM Method 8318. The urine was obtained 

from personnel in the Taft building at NIOSH and then combined and mixed in the BHAB 

labs into a single pool of urine from which all samples were prepared. A total of 25 urine 

samples were prepared. Five samples were left blank. Five samples were prepared containing 

the analyte at each of the following levels: 2.02 mg/L, 4.09 mg/L, 49.3 mg/L, and 143.6 

mg/L. The samples were prepared and shipped frozen to ALS Environmental on April 7, 

2015 and arrived there the next day. The samples were analyzed on April 10, 2015. The 

sample preparation procedure and analytical conditions found in draft method 8318 were 

used without exception. 

For this analysis, the Lower Limit of Quantitation (LLOQ) was determined by ALS to be 

0.70 mg/L for the compound of interest. As mentioned above, the spike levels ranged from 

2.02 to 143.6 mg/L, which is 3 to 200 times the LLOQ and fall within the method detection 

range of 0.2 to 212 mg/L. 

The table (Table 1) below shows the data obtained from the User Check samples. 2,5-

Hexanedione was not detected in any of the blank samples (data not shown), so no 

corrections were required. A summary table (Table 2) of average recoveries and precision as 

calculated by relative standard deviation (RSD) follows.
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

Table 1

Spike 

ID

Target concentration 

(mg/L)

Concentration found 

(mg/L)

Recovery 

(%)

5 2.02 1.80 89.11

11 2.02 1.90 94.06

16 2.02 0.37 18.32

20 2.02 1.84 91.09

24 2.02 1.88 93.07

4 4.09 3.78 92.42

8 4.09 3.71 90.71

15 4.09 4.00 97.80

18 4.09 3.92 95.84

21 4.09 3.88 94.87

1 49.3 48.1 97.57

2 49.3 48.6 98.58

10 49.3 49.9 101.22

13 49.3 48.3 97.97

22 49.3 48.8 98.99

3 144 134 93.06

7 144 136 94.44

12 144 126 87.50

14 144 174 120.83

25 144 132 91.67

Table 2

Spiked amount mg/L Recovery (%) RSD (%)

2.02 77.1 42.7

4.09 94.3 2.97

49.3 98.9 1.44

143.6 97.5 13.6

Overall 92.0 20.3

Statistical tests for outlier points (Dixon’s Q-test and Grubbs test) were performed on the 

data at each concentration level. No outliers were found at the three highest concentration 

levels, but one sample in the lowest concentration level, Sample 16 (18.32%), was 

determined to be an outlier by both statistical tests. Table 3 gives the summary values for 

accuracy and precision when this rejected data point has been removed. 
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

Table 3

Spiked amount mg/L Recovery (%) RSD (%)

2.02 91.8 2.39

4.09 94.3 2.97

49.3 98.9 1.44

143.6 97.5 13.6

Overall 95.8 7.37

Once this outlier is removed, the accuracy at every concentration level is within ± 9% of the 

true value, which is well within acceptable values for biological monitoring methods. The 

relative standard deviation (RSD) for each individual level ranges from 1 to 14 per cent. 

These precision values (and the overall precision) are also well within acceptable limits. Two 

of the primary guidelines on bioanalytical method validation state that accuracy and precision 

should be within ± 15% at each level and within ± 20% at the lowest level [1,2]. The contract 

lab reported no difficulties understanding the draft method nor in setting it up or analyzing 

the samples. The method has relatively few analytical steps, is quite straightforward, is 

sensitive enough to determine occupational exposures, and has been shown to have adequate 

precision and accuracy. It is recommended that the method, NMAM Method 8318 (2,5-

hexanedione in urine) be approved and accepted for inclusion in the NIOSH Manual of 

Analytical Methods. 

Dale Shoemaker, PhD 

Research Chemist

November 9, 2015 

References

[1] European Medicines Agency [2011]. Guideline on bioanalytical method validation 

[http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/

08/WC500109686.pdf]. 

[2] FDA [2001]. Guidance for industry. Bioanalytical method validation. 

[http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf].