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These procedures were designed and tested for internal use by OSHA personnel. Mention of any company name or commercial product does not constitute endorsement by OSHA.
||0.25 ppm (1 mg/m3)
||Samples are collected by drawing air through glass fiber filters coated with 2 mg of 3,4-dimethoxybenzylamine
(veratrylamine). Samples are extracted with 90:10 (v/v) acetonitrile/dimethylsulfoxide and
analyzed by HPLC using a UV detector. (Filters as prepared in Methods 98 and 102 can also
be used for this procedure.)
|Recommended air volume
and sampling rate:
|60 L at 0.5 L/min
|Reliable quantitation limit:
||8.3 ppb (33 µg/m3)
|Standard error of estimate
at the target concentration:
||Submit the samples for analysis as soon as possible after sampling. If delay is
unavoidable, store the samples in a refrigerator. Store samples in a refrigerator upon
receipt at the laboratory.
|Status of method:
||Evaluated method. This method has been subjected to the established evaluation
procedures of the Organic Methods Evaluation Branch.
|Date: December 1990
||Chemist: Yihlin Chan
Organic Methods Evaluation Branch
OSHA Analytical Laboratory
Salt Lake City, Utah
1. General Discussion
In OSHA Method 25 (Ref. 5.1.), maleic anhydride is
collected and derivatized on p-anisidine-coated XAD-2 tubes. An
untreated XAD-2 tube is connected in series to trap the p-anisidine
that is partly leached from the first tube during sampling. In trying to develop sampling
and analytical methods for a series of anhydrides (acetic, maleic, phthalic, and
trimellitic), derivatizing agents other than p-anisidine were investigated to
obviate the use of the second tube. 1-(2-Pyridyl)piperazine, the agent used
in earlier methods for a series of isocyanates (Refs. 5.2.-5.4.), was considered. A sampling method for acetic
anhydride was validated using this derivatizing agent (Ref.
5.5.). With maleic and other anhydrides, however, this reagent was found to be
unsatisfactory due to the instability of its derivatives. After a brief survey,
veratrylamine was selected because: (1) it readily forms relatively stable acid-amides
with these anhydrides, (2) it does not leach from the glass fiber filters impregnated with
it, even at a flow of 1 liter per minute, and (3) its cost is reasonable. The sampling
device consists of two filters which are separated by a center support section (Figure 2.1.2.). Samples are collected closed-face to
1.1.2. Toxic effects (This section is for information only and should not be taken as
the basis of OSHA policy.)
Inhalation of subacute levels of maleic anhydride can cause severe headaches,
nosebleeds, nausea, and temporary impairment of vision. Exposure to maleic anhydride can
also lead to conjunctivitis and corneal erosion. Repeated exposure to concentrations above
1.25 ppm has caused asthmatic responses in workers. Allergies have developed so that lower
concentrations of maleic anhydride can no longer be tolerated. An increased incidence of
bronchitis and dermatitis has also been noted among workers with long-term exposure to
maleic anhydride. One case of pulmonary edema in a worker was reported. (Ref. 5.6.)
1.1.3. Workplace exposure
Through Diels-Alder syntheses and co-polymerization reactions, maleic anhydride is used
in the manufacture of alkyd-type resins, dye intermediates, pharmaceuticals,
agricultural chemicals, maleic hydrazide, malathion, and others. (Ref. 5.7.)
According to the 1972 NIOSH National Occupational Exposure Surveys, exposure tomaleic
anhydride was noted in the following industries: nonmetallic mineral products (SIC code
3299), percent of employee exposed, 30.2%; terrazzo, tile, marble, mosaic (1743), 19.4%;
printing ink (2893), 19.1%; paints and allied products (2851), 16.0%; transportation
equipment and supplies (5088), 14.3%; bags other than textile bags (2643), 8.6%; and metal
cans (3411), 6.9%. The 1982 NIOSH surveys found 28.1% of the employee in the adhesives and
sealants (SIC code 2891) exposed to maleic anhydride. (Ref.
1.1.4. Physical properties and other descriptive information (Ref. 5.9. unless noted otherwise)
||2,5-furandione; cis-butenedioic anhydride; dihydro-2,5-dioxofuran; maleic
acid anhydride; toxilic anhydride
||<21.3 Pa (0.16 mmHg) at 25°C
||soluble in water, acetone, alcohol, and dioxane; partially soluble in chloroform and
|Derivative (Ref. 5.10.)
||white crystalline solid
||soluble in chloroform, methanol, acetonitrile, dimethylsulfoxide (DMSO).
The analyte air concentrations throughout this method are based on the recommended
sampling and analytical parameters. Air concentrations listed in ppm are referenced to
25°C and 101.3 kPa (760 mmHg). The analyte concentrations are listed as those of maleic
anhydride even though the derivative is the actual species analyzed.
1.2. Limit defining parameters
1.2.1. Detection limit of the analytical procedure
The detection limit of the analytical procedure is 7.1 ng per injection (15 µL
injection of 0.471 µg/mL solution). This is the amount of analyte which gave a peak whose
height is approximately 5 times the baseline noise. (Section
1.2.2. Detection limit of the overall procedure
The detection limit of the overall procedure is 1.99 µg per sample (8.3 ppb, 33 µg/m3).
This is the amount of analyte spiked on the sampling device which allows recovery of an
amount equivalent to the detection limit of the analytical procedure. (Section 4.2.)
1.2.3. Reliable quantitation limit
The reliable quantitation limit is 1.99 µg per sample (8.3 ppb, 33 µg/m3).
This is the smallest amount of analyte spiked on the sampling device which can be
quantitated within the requirements of a recovery of at least 75% and a precision (±1.96
SD) of ±25% or better. (Section 4.3.)
The reliable quantitation limit and detection limits reported in the method are based
upon optimization of the instrument for the smallest possible amount of the analyte. When
the target concentration of the analyte is exceptionally higher than these limits, they
may not be attainable at the routine operating parameters.
1.2.4. Instrument response to the analyte
The instrument response over the concentration range of 0.5 to 2 times the target
concentration is linear. (Section 4.4.)
The recovery of the maleic anhydride-veratrylamine derivative (MAVA) from the samples
used in a 15-day storage test remained essentially 100% when the samples were
stored at 0°C. The recovery was 72% after 15 days when the samples were stored at ambient
temperature. (Section 4.5.)
1.2.6. Precision (analytical procedure only)
The pooled coefficient of variation obtained from replicate injections of analytical
standards at 0.5, 1, and 2 times the target concentration is 0.0074. (Section 4.6.)
1.2.7. Precision (overall procedure)
The precision at the 95% confidence level for the refrigerated 15-day storage test is
±17.4%. (Section 4.7.) This includes an additional ±5%
for pump error. The overall procedure must provide results at the target concentration
that are ±25% or better at the 95% confidence level.
A draft copy of this procedure and six samples collected from a controlled test
atmosphere (80% RH) were given to a chemist unassociated with this evaluation. The samples
were analyzed after having been stored in a refrigerator at 0°C for 4 days. None of the
sample results differed from its theoretical value by more than the precision reported in Section 1.2.7. (Section 4.8.)
1.3.1. The maleic anhydride is derivatized in situ eliminating the possibility
of its being hydrolyzed during storage. Interference from maleic acid is also eliminated.
1.3.2. Because the derivatizing agent is not leached from the filter during sampling,
there is no need for an additional sorbent tube downstream of the sampler.
The sampling medium is not available commercially.
2. Sampling Procedure
2.1.1. A personal sampling pump that can be calibrated to within ±5% of the
recommended flow rate with the sampling device in line.
2.1.2. A three-piece polystyrene cassette containing two glass fiber filters, each
coated with 2 mg of veratrylamine. (Figure 2.1.2.) Coated
filters are prepared by applying 0.5 mL of a solution of 4 mg/mL veratrylamine in
methylene chloride to each glass fiber filter and allowing them to dry in a hood or under
vacuum. Store the coated filters in a closed jar in a refrigerator and use them within a
month after preparation.
No reagent is required for sampling.
2.3. Sampling technique
2.3.1. Remove the end plugs from the inlet and the outlet of the sampler. Attach the
sampler to the sampling pump with a piece of flexible tubing and place it in the worker's
2.3.2. Replace the end plugs after sampling. Seal the sample end-to-end
with an official OSHA seal (Form 21).
2.3.3. Submit at least one blank with each set of samples. Handle the blank the same as
the other samples except draw no air through it.
2.3.4. List any potential interferences on the sample data sheet.
2.3.5. Submit the samples to the laboratory for analysis as soon as possible after
sampling. If delay is unavoidable, store the samples at reduced temperature.
2.4. Sampler capacity
The sampler capacity was evaluated with a test atmosphere (80% RH) at 1.9 times the
target concentration. The sampler capacity exceeds 130 L. (Section
2.5. Extraction efficiency and stability of extracted samples (Section 4.10.)
2.5.1. The average extraction efficiency at the target concentration was essentially
2.5.2. Extracted samples remain stable for at least 3 days when stored at room
2.6. Recommended air volume and sampling rate
2.6.1. The recommended air volume is 60 L.
2.6.2. The recommended air sampling rate is 0.5 L/min.
2.7. Interferences (sampling)
Excessive amounts of compounds that can react with veratrylamine, such as isocyanates,
acid chlorides, and anhydrides other than maleic, may reduce the sampler capacity by
consuming part of the derivatizing agent.
2.8. Safety precautions (sampling)
Attach the sampling equipment to the worker in such a manner that it will not interfere
with work performance or safety. Follow all safety practices applicable to the work area.
3. Analytical Procedure
3.1.1. An HPLC equipped with a UV detector. A Waters 600E pump, a 900 photodiode array
detector, and a WISP autosampler were used in this evaluation.
3.1.2. An HPLC column capable of separating veratrylamine, MAVA, and any interferences.
An Alltech C8 column (4.6 mm × 25 cm, 10-µ particle size) was used in this
3.1.3. An electronic integrator or other suitable means of measuring detector response.
A Hewlett-Packard 3357 laboratory data system was used in this evaluation.
3.1.4. Sample vials, 4-mL glass, with Teflon-lined septum caps. WISP vials were used in
3.1.5. Volumetric flasks and pipets.
3.1.6. A mechanical shaker. A Fisher Roto-Rack® was used in this
3.2.1. Maleic anhydride-veratrylamine derivative (MAVA). Synthesized as in Section 4.12.
3.2.2. Dimethyl sulfoxide (DMSO). Dimethyl sulfoxide was obtained from Baxter Burdick
3.2.3. Acetonitrile. Acetonitrile was obtained from Baxter Burdick and Jackson.
3.2.4. Extraction solvent, acetonitrile/DMSO 90:10 (v/v).
3.2.5. Phosphoric acid. Phosphoric acid was obtained from J T Baker.
3.2.6. Water, HPLC grade. The water was from an in-house Millipore milli-Q
water purification system.
3.3. Standard preparation
3.3.1. Prepare stock standards by weighing 10-20 mg of MAVA in 10-mL
volumetric flasks and diluting to volume with the extraction solvent. Apply a factor of
0.3697 to the weight of MAVA to convert it to that of free maleic anhydride. For example,
10 mg of MAVA dissolved in 10 mL will give a standard stock solution representing 0.3697
mg/mL or 369.7 µg/mL of maleic anhydride.
(MW maleic anhydride) / (MW MAVA) = 98.06 / 265.27
3.3.2. Prepare analytical standards by further diluting the stock standards with the
extraction solvent. An analytical standard of 15 µg/mL represents 1 times the target
3.3.3. Prepare a sufficient number of standards to generate calibration curves.
Analytical standard concentrations must bracket sample concentrations.
3.4. Sample preparation
3.4.1. Transfer the front and the back filters to separate WISP vials. This is best
accomplished by double-folding the filter with the folds parallel.
3.4.2. Add 4.0 mL of the extraction solvent to each vial.
3.4.3. Cap the vials and shake them on a mechanical shaker for 1 h.
3.5.1. HPLC conditions
||Alltech C8 (4.6 mm × 25 cm, 10-µ particle size)
||acetonitrile/water/phosphoric acid 30:70:0.1 (v/v/v)
||254 nm (This wavelength was used because it gave good resolution of analyte from other
components and good sensitivity.)
3.5.2. Measure detector response using a suitable method such as electronic
3.5.3. Construct a calibration curve using an external standard method by plotting µg
per sample versus detector response of standard injections.
3.6. Interferences (analytical)
3.6.1. Any compound that absorbs at 254 nm and has a similar retention time as MAVA is
a potential interference. Generally, chromatographic conditions can be altered to separate
3.6.2. Retention time on a single column is not considered proof of chemical identity.
Additional means of identification include: analysis using an alternate HPLC column,
detection at another wavelength, and comparison of absorbance response ratios.
The analyte amount for samples is obtained from the calibration curve in terms of
micrograms per sample uncorrected for extraction efficiency. The analyte amount is
corrected by subtracting the amount found in the blank. The air concentration is obtained
by using the following equations.
(liters of air sampled)(extraction efficiency)
||molar volume (liters) at 101.3 Pa (760 mmHg) and 25°C
||molecular weight of maleic anhydride
3.8. Safety precautions (analytical)
Avoid skin contact and inhalation of all chemicals. Restrict the use of all chemicals
to a fume hood when possible. Wear safety glasses and a lab coat at all times while in the
4. Backup Data
4.1. Detection limit of the analytical procedure
The detection limit of the analytical procedure is 7.1 ng (15-µL
injection of a 0.471 µg/mL solution). This is the amount of analyte that will give a peak
with height approximately 5 times the height of the baseline noise. A chromatogram of the
detection limit of the analytical procedure is shown in Figure
4.2. Detection limit of the overall procedure
The detection limit of the overall procedure is 1.99 µg per sample (8.3 ppb, 33 µg/m3).
This is the amount of analyte spiked on the sampling device which allows recovery of an
amount equivalent to the detection limit of the analytical procedure. Six veratrylamine-coated
glass fiber filters were each liquid spiked with 1.99 µg of maleic anhydride (20 µL of a
99.6 µg/mL solution). The samples were stored at room temperature and extracted 24 h
later with 4.0 mL of the extraction solvent. The injection size listed in the analytical
procedure (15 µL) was used in the determination of the detection limit of the overall
Detection Limit of the Overall Procedure
4.3. Reliable quantitation limit
The reliable quantitation limit is also 1.99 g per sample (8.3 ppb, 33 µg/m3).
This was derived from the samples and data of Table 4.2. Because the recovery was greater
than 75% and the precision (±1.96 SD) was ±25% or better, the detection limit of the
overall procedure and reliable quantitation limit are the same.
Reliable Quantitation Limit
(based on samples and data of Table 4.2.)
4.4. Instrument response
The instrument response to MAVA over the range of 0.5 to 2 times the target
concentration is linear with a slope of 58352 area counts per microgram per sample. The
responses to MAVA at 254 nm were determined by multiple injections of analytical
standards. The data which are summarized in Table 4.4. are presented graphically in Figure 4.4.
Instrument Response to MAVA
|× target concn
4.5. Storage data
A test atmosphere (80% RH) containing 1.86 mg/m3 of maleic
anhydride was prepared in a vapor generator. Thirty-six samples were
collected at 0.5 L/min for 60 min. Six samples were analyzed immediately after the
generation. Fifteen samples were stored in a refrigerator (0°C) and the other fifteen
were stored in the dark at ambient temperature (20-25°C). Every few days
over a 15-day period, three samples were selected from each of the two sets
and analyzed. The results are listed in Table 4.5. and presented graphically in Figures 4.5.1. and 4.5.2.
4.6. Precision (analytical procedure)
The precision of the analytical procedure is 0.0074. The precision of the analytical
procedure is defined as the pooled coefficient of variation determined from multiple
injections of analytical standards representing 0.5, 1, and 2 times the target
concentration (Section 4.4.).
Precision of the Analytical Procedure
(based on the data of Table 4.4.)
|× target concn
4.7. Precision (overall procedure)
The precision of the overall procedure is determined from the storage data. The
determination of the standard error of estimate (SEE) for a regression line plotted
through the graphed storage data allows the inclusion of storage time as one of the
factors affecting overall precision. The SEE is similar to the standard deviation except
it is a measure of dispersion of data about a regression line instead of about a mean. It
is determined with the following equation:
|total no. of data points
2 for linear regression
3 for quadratic regression
||observed % recovery at a given time
||estimated % recovery from the regression line at the same given time
An additional ±5% for pump error is added to the SEE by the addition of variances. The
precision at the 95% confidence level is obtained by multiplying the SEE (with pump error
included) by 1.96 (the z-statistic from the standard normal distribution at
the 95% confidence level). The 95% confidence intervals are drawn about their respective
regression lines in the storage graphs as shown in Figure
4.5.1. The precision of the overall procedure of ±17.4% was obtained from Figure 4.5.1.
4.8. Reproducibility data
Six samples, collected at 0.5 L/min for 60 minutes from a controlled test atmosphere
[80% RH, 20-25°C, 87.7 kPa (658 mmHg)] containing 2.1 mg/m3
of maleic anhydride, were given to a chemist unassociated with this evaluation. The
samples were stored for 4 days at about 0°C before being analyzed. No sample result had a
percent deviation greater than the precision of the overall procedure.
4.9. Sampler capacity
Sampler capacity was tested by sampling a test atmosphere of 1.9 mg/m3
maleic anhydride at ambient temperature and 80% relative humidity. Two samplers were
placed in series. The upstream sampler contained only one filter. The back sampler was
replaced with a new one every 40 minutes to monitor the downstream air concentration. The
sampling rate was 0.501 L/min. Breakthrough was not seen in 280 minutes of testing.
Breakthrough Data at 1.9× Target Concentration
4.10. Extraction efficiency and stability of extracted
4.10.1. Extraction efficiency
The extraction efficiency for MAVA was determined by analyzing the veratrylamine-impregnated
glass fiber filters that had been liquid-spiked with maleic anhydride at the
target concentration. These samples were stored at ambient temperature overnight before
the extraction and analysis. The average extraction efficiency was 97.8%.
4.10.2. Stability of the extracted samples
The stability of the extracted samples was investigated by reanalyzing the extracted
samples with fresh standards 3 days after the original analysis. The samples had been
recapped and stored at room temperature. The change in the recovery averaged -0.9%.
Stability of Extracted Samples
3 days (%)
A chromatogram at the detection limit of the analytical procedure is shown in Figure 4.1. and a chromatogram of one of the 15-day
refrigerated samples is shown in Figure 3.5.1.
4.12. Synthesis of MAVA
Veratrylamine, 97%, from Aldrich
Maleic anhydride, from Aldrich
Toluene, from Baxter Burdick and Jackson
Isooctane, from Fisher Scientific
Chloroform, from Fisher Scientific
Fritted-glass filtering funnel
Explosion-proof hot plate
Recrystallize the maleic anhydride from toluene. Add 0.98 g (0.01 mole) of maleic
anhydride to a solution of 1.67 g (0.01 mole) of veratrylamine in 10 mL of chloroform.
Stir the mixture for 10 min. Evaporate the chloroform in a hood. Dissolve the residue in a
minimal amount of chloroform. While heating on a hot plate, slowly add isooctane until the
solution just becomes cloudy. Clear the solution with an addition of a drop of chloroform.
Remove from the hot plate. After the solution has cooled to room temperature, set in a
freezer overnight. Collect the crystals that formed. The melting point should be 146.0-146.5°C.
The yield should be quantitative.
Figure 1.1.4. UV spectrum (in water/acetonitrile/phosphoric acid = 70:30:0.1 (v/v/v)) and molecular structure of MAVA.
Figure 2.1.2. A drawing of a sampling device for maleic anhydride.
Figure 3.5.1. Chromatogram at target concentration. 1 = veratrylamine, 2 = MAVA.
Figure 4.1. Detection limit of the analytical procedure. 1 = MAVA.
Figure 4.4. Calibration curve for MAVA.
Figure 4.5.1. Storage test at reduced temperature.
Figure 4.5.2. Storage test at ambient temperature.
5.1. "OSHA Analytical Methods Manual", Second Edition,
U.S. Department of Labor, Occupational Safety and Health Administration; OSHA Analytical
Laboratory: Salt Lake City, UT, 1990; Method 25; American Conference of Governmental
Industrial Hygienists (ACGIH): Cincinnati, OH, Publication No. 4542.
5.2. ibid. Method 42.
5.3. ibid. Method 47.
5.4. ibid. Method 54.
5.5. Chan, Y., "OSHA Method 82: Acetic Anhydride", OSHA
Analytical Laboratory, unpublished, Salt Lake City, UT 84165, April 1990.
5.6. Sittig, M., "Handbook of Toxic and Hazardous
Chemicals", Second Edition, Noyes Publications, Park Ridge, NJ, 1985.
5.7. Budavari, S., Ed., "Merck Index", Eleventh Edition,
Merck & Co., Rahway, NJ, 1989.
5.8. "OSHA Regulated Hazardous Substances, Industrial Exposure
and Control Technologies", U.S. Department of Labor, Occupational Safety and Health
Administration, Washington, D.C., 1990, ISBN: 0-86587-795-5.
5.9. Sweet, D.V., Ed., "Registry of Toxic Effects of Chemical
Substances", 1985-86 edition, U.S. Department of Health and Human
Services, Government Printing Office, DHHS(NIOSH) Publication No. 87-114.
5.10. Author's personal observation.