Issued: 4/15/00

Revised 02/16/04

Analysis of Isocyanates
Liquid Chromatography  Diode Array/MSD

1.0 Scope and Application

1.1 This method is applicable for the determination of isocyanates prepared by derivatization of analytes extracted
from solids, soils, filters, and foams or collected in impinger solutions. The following isocyanates, may be determined
by this method: 2,4-toluene diisocyanate (CAS 584-84-9), 2,6-toluene diisocyanate (CAS 91-08-7),
1,6-hexamethylene diisocyanate (CAS 822-06-0), 1,6-hexamethylene diisocyanate biuret (CAS 4035-89-6), 1,6-
hexamethylene diisocyanate trimer (CAS 28182-81-2, isophorone diisocyanate (CAS 4098-71-9), 2,4’-methylene
diphenyl diisocyanate (CAS 5873-54-1) and 4,4’-methylene diphenyl diisocyanate (CAS 101-68-8).

1.2 This method is restricted to use by, or under the direct supervision of analysts experienced in chromatography
and the interpretation of chromatographic results. Each analyst must demonstrate the ability to generate acceptable
results with this method.

1.3 The toxicity of each reagent has been precisely defined. The exposure to these chemicals must be reduced to the
lowest possible level by whatever means available. The laboratory maintains a current awareness file of OSHA
regulations regarding safe handling of the chemicals specified in this method. A reference file of material safety data
sheets is available to all personnel involved in chemical analysis. Refer to the Chemical Hygiene Plan for additional
information regarding laboratory safety.

2.0 Summary of Method

2.1 The extract obtained from the solid sample, i.e. soil, foam, etc., is evaporated to dryness and re-dissolved in
90:10 Acetonitrile:Dimethyl sulfoxide [TDI,MDI] or 70:30 Acetonitrile:Dimethyl sulfoxide [HDI, IPDI].

2.2 Samples obtained from impingers are evaporated to dryness and re-dissolved in the appropriate
Acetonitrile:Dimethyl sulfoxide solution.

2.3 Filter samples which have been field desorbed with Acetonitrile:Dimethyl sulfoxide are analyzed directly. If other
solvents are used, the extract is evaporated to dryness and re-dissolved in the appropriate Acetonitrile:Dimethyl
sulfoxide solution.

3.0 Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample
processing hardware. All of these materials must be routinely demonstrated to be free from interferences under the
conditions of the analysis by preparing and analyzing laboratory method (or reagent) blanks.

3.1.1 Glassware must be thoroughly cleaned prior to use.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems in sample analysis.

3.2 Compounds existing in the sample matrix that pass through the LC column and have an absorbance at the
monitored wavelength may be a chemical interference. Changing the chromatographic conditions may allow a
separation of peaks suitable for determination of the isocyanates. It is especially important in these cases to confirm
the positive identification of isocyanates with matrix fortified samples.

3.3 Under the chromatographic conditions, HDI monomer and 2,6-TDI co-elute; consequently, if the two compounds

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Revised 02/16/04

are thought to be present then a mass selective detector must be employed.

4.0 Apparatus and Materials

4.1 High Performance Liquid Chromatograph (HPLC):

4.1.1 Hewlett Packard HP-1100 LC, equipped with a Diode Array and MS detectors.

4.1.2 Phenomenex Luna 3µ C8(2) 100Å 100 X 2.00 mm, Analytical Part No. 00D-42486-B0

4.1.3 Phenomenex Security Guard Cartridge 4 X 2.0 mm Part No. AJO-4350

4.2 Vials, 20-mL with Teflon®-lined caps.

4.3 Volumetric flask, 5-mL.

4.4 Analytical balance: Capable of weighing to the nearest 0.1 mg.

5.0 Reagents

5.1 Water, Fisher Scientific Optima grade, Cat. No. W7-4, or equivalent: Water must be free of interferences at the
IDL for each analyte.

5.2 Acetonitrile, Fisher Scientific HPLC grade, Cat. No. A998-4, or equivalent.

5.3 Methylene chloride, Fisher Scientific Optima grade, Cat. No. D151-4, or equivalent.

5.4 Hexane, Fisher Scientific Optima grade, Cat. No. H303-1, or equivalent.

5.5 Ammonium acetate, Aldrich Cat. No. 24,019-2.

5.6 Acetic acid, glacial, Fisher Scientific Cat. No. A38S-212.

5.7 1-(2-Pyridyl)piperazine (1,2-PP) solution, Aldrich Cat. No. 15,127-0.

5.8 1-(2-Methoxyphenyl)piperazine (1,2-MP, 1,2-MOPP) solution, Aldrich Cat. No. M2,260-1

5.9 Ammonium acetate buffer solution (AAB), 0.1 M: Prepared by weighing out 7.705 g of ammonium acetate and
diluting to 1 L with HPLC grade water. Adjust the pH to 6.2 with glacial acetic acid.

5.10 Dimethyl sulfoxide, Baxter HPLC grade, Cat. No. 081-1.

6.0 Sample Collection, Preservation, and Handling

6.1 Samples must be stored at 4°C prior to analysis. Samples must be concentrated within 30 days of extraction and
should be analyzed as soon as possible.

7.0 Procedure

7.1 Sample Preparation:

7.1.1 Samples that are soils, solids, or foams are extracted using the Accelerated Solvent Extractor or Soxhlet
technique.

7.1.1.1 Extract 10.0 g of sample using standard Soxhlet procedures except, 40 mg of 1,2-PP must be added
to the methylene chloride in the receiving flask. Derivatization of the isocyanate will occur during extraction.
In the case of the ASE, the reagent, dissolved in 2 mL of solvent, is added to the receiving vessel during the
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Revised 02/16/04

set up of the extractor.

7.1.1.2 Quantitatively transfer the extract to a 5-mL volumetric flask and gently evaporate to dryness using a
stream of nitrogen.

7.1.1.3 Re-dissolve the extract in the appropriate Acetonitrile:Dimethyl sulfoxide solution and dilute to the
mark.

7.1.2 Toluene impinger samples should be received with the isocyanates already derivatized and in toluene. The
sample should be evaporated to dryness and redissolved in the appropriate Acetonitrile:Dimethyl sulfoxide
solution. To insure that all of the toluene has been removed the following solvent exchange procedure is
recommended.

7.1.2.1 Solvent Exchange

The impinger sample (in its entirety) is placed in a concentrator vessel (example: Kuderna-Danish, Rapid
Vap) and concentrated to less than 2 mL (less than 1 mL is preferable for toluene) with the settings for the
concentrator as follows:

TEMPERATURE SETTINGS – 70 DEGREES C

TEMPERATURE ACTUAL – 65 DEGREES C
VORTEX ACTION – 90
TIME SETTINGS – 15 min intervals (~4-5 will be followed)

Add 50 mL of a 95:5 acetonitrile: dimethyl sulfoxide mixture directly in to the vessel. Gently swirl the
concentrator tube so that the 95:5 mixes in its completely with the small amount of sample left in the
concentrator vessel. Place the vessel in the concentrator and resume concentrating at the same settings as
before until the extract reaches <1 mL or near dryness.

Qualitatively transfer the sample into a 5 mL volumetric flask* with the appropriate Acetonitrile:Dimethyl
sulfoxide solution and mixed.

Transfer 1-2 mL of the final sample into a 2-mL autosampler vial and store the vial at 4° C until analysis is
performed.

* If samples are known to be very low in concentration or if a very low MDL is necessary, a smaller final
volume maybe necessary.

7.1.3 Filter samples are desorbed in the field by addition of either 5 mL of the appropriate Acetonitrile:Dimethyl
sulfoxide solution and are ready for analysis. If other solvents are used for field desorption, the extract is
evaporated to dryness and re-dissolved in the appropriate Acetonitrile:Dimethyl sulfoxide solution. The volume
used for desorption is dictated by the concentration expected.

7.2 Instrument Conditions:

7.2.1 Fill the reservoirs with the solvents listed below:

Pump B acetonitrile (section 5.2)

Pump C ammonium acetate buffer (section 5.9)

7.2.2 Install the LC column taking care not to overtighten the plastic fittings (fingertight is sufficient). A guard
column, complementary to the column, should be installed prior to the analytical column.

7.2.3 Start the pumps and allow the column to equilibrate for 30 min. Make sure all fittings are leak-free.

7.2.4 Set the liquid chromatograph and detectors to the following parameters:

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Pump:
Stop Time 20 min
Post Time 5.00 min
Flow 300 µL/min
Oven 40°C
Temperature
Injector:
Injection Volume 5.0 µL Draw Speed 200 µL/min
Diode Array Detector Settings:
Stop Time as Pump: 20.00 min
Post Time Off
Peak Width 0.050 min
Sampling 0.320 s
Autobalance On
Signals:
Sample, A 254 nm
Sample, C 240 nm
Stored Yes
Band Width 4 nm
Reference C 490 nm
Band Width 40 nm
Spectrum:
Store All in Peak
From 220 nm
To 500 nm
Step 2 nm
Threshold 1.0 mAU
UV Lamp required Yes
VIS Lamp required No
Autobalance Prerun No
Autobalance Postrun No

MSD Scan
Stop Time as Pump: 20.00 min
APCI Positive
Mass Range Varies with compound of
interest
Gas Temperature 300°C
Vaporizer Temp. 350°C
Drying Gas 6.0 L/min
Neb. Pressure 60 psig
Vcap 2500 V
Cornona 4.0 µA
Data Storage Condensed
Fragmentor Disable Ramp

MSD SIM Parameters

Group Name Sim Ions Fragmentor
TDI-PP 501.40, 338.1 150
HDI-PP 495.00 150
HDI Biuret-PP 968.7, 805.5 150
HDI Trimer-PP 994.5, 831.5 150
IPDI-PP 549.3 150
MDI-PP 577.00 150
Gain EMV – 3.0 SIM Resol – Low Polarity - Positive Actual Dwell - 229

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 Issued: 4/15/00
Revised 02/16/04

Time Table:
Time [min] %B %C mL/min
0 35 65 0.300
0.5 35 65 0.300
10.00 70 30 0.300
11.00 70 30 0.300
16.01 35 65 0.300

7.3 Calibration:

7.3.1 Establish the retention times for each of the isocyanates of interest using the chromatographic conditions
provided above. The retention times shown in Table 1 are to be used as guidance only.

7.3.2 Make suitable dilutions of the isocyanate-1, 2-PP derivative standard solutions to calibrate the LC.
Suggested concentrations are 0.5, 1, 5, 10, 20,and 50 ng/µL.

7.3.3 Analyze 5.0 µL of each calibration standard and record the area of each peak and the concentration of the
compound. Figure 1 shows a chromatogram of a typical mixed isocyanate-PP standard.

7.3.4 The ChemStation will construct a calibration curve for each analyte. The calibration is acceptable if the r2
value is greater than 0.99 (correlation coefficient r > 0.995).

7.3.5 The ChemStation calibration curves must be set to linear with forced origin on the MSD section in order to
obtain the required LOQ.

7.4 Continuing Calibration:

7.4.1 A 10.0-ng/µL calibration check solution must be analyzed every 12 hours or after 20 samples. The response
of each compound must be within ± 10% from the response predicted by the calibration curve or else that
parameter is considered to be out of control.

7.4.2 If a parameter is out of control, check the HPLC system and correct any problems, prepare and analyze a
fresh calibration check solution, or clean the detector.

7.4.3 If the parameter is still out of control, prepare and analyze a new five-point calibration.

7.5 HPLC Analysis:

7.5.1 Analyze the samples using the HPLC conditions listed in section 7.2.4.

7.5.2 If the response for any of the isocyanate ureas exceeds the initial calibration range of the HPLC system, the
sample must be reanalyzed following dilution with the appropriate Acetonitrile:Dimethyl sulfoxide solution.

7.5.3 Samples containing <1 µg/mL of the analyte should be quantified using MSD data.

7.6 Calculations:

7.6.1 The concentration of each isocyanate urea is given directly in the sample run report printed by the
ChemStation based upon the current calibration data for that analyte. The value is usually given in ng/µL.

7.6.2 Convert the result to concentration of the underivatized isocyanate using the following equation:

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 Issued: 4/15/00
Revised 02/16/04

C Iso-Urea * MW Iso
CIso, ng/µL =
MW Iso-Urea

Where,

C Iso-Urea = the concentration of the derivatized isocyanate from the sample run report,

MW Iso = the molecular weight of the free isocyanate (from Table 1), and

MW Iso-Urea = the molecular weight of the derivatized isocyanate (Table 1).

7.6.3 Multiply the value by the dilution factor, if necessary. This dilution factor must take into account changes in
volume of the solution during preparation, unless included elsewhere in the calculations.
7.6.4 The concentration should be converted for solids as follows:

C Iso * 5.0 mL
C Iso, mg/Kg =
Ws

Where,

Ws = the dry weight of the sample in grams (from SP-3).

8.0 Quality Control

8.1 Required instrument QC is found in the following sections:

8.1.1 There must be an initial calibration of the HPLC/DAD system as specified in section 7.3.

8.12 The HPLC/DAD system must meet continuing calibration criteria each 12 hr as specified in section 7.4.

8.2 The laboratory will on an ongoing basis analyze a reagent blank, a matrix spike, and a matrix spike duplicate for
each analytical batch to assess accuracy.

8.2.1 The concentration of the matrix spike is generally the level of the mid-range standard.

8.2.2 The accuracy is defined as the percent recovery of the matrix spike analyses. The percent recovery should
be within the range 80 - 120%, or else a matrix interference will be suspected.

8.2.3 The relative percent difference of the duplicates should be no greater than ±25%.

9.0 Method Performance

9.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured
and reported with 99% confidence that the value is above zero. The MDL concentrations are obtained for each
analyte by measuring seven replicates of a low-level solution brought through the entire analytical procedure. The
MDL is three times the standard deviation of the measured value for each analyte. The MDLs are obtained on at least
a yearly basis, and the most recent determinations are listed in Table 1.

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Table 1 Typical Retention Times (DAD/MSD) and IDLs

Parameter Retention Time* IDL** MWISO MWISO-PP Urea Monitoring Ion
1,6-HDI 3.098/3.417 0.0060(0.00215) 168.0 494.4 495
HDI Biuret 8.21/89.38 0.049(0.024) 478 967 968.7/805.5
HDI Trimer 9.14/9.34 0.037(0.015) 504.5 993.5 994.5/831.5
IPDI (2 isomers) 5.20(7.09)/5.39(7.29) 0.033(0.0135) 222.3 548.3 549.3
2,4-TDI 3.669/4.021 0.003(0.0010) 174.2 500.6 501
2,6-TDI 3.098/3.417 0.005(0.0017) 174.2 500.6 501
4,4'-MDI 6.692/7.044 0.073(0.0318) 250.3 576.7 577
2,4’-MDI 5.908/6.239 0.073(0.0318) 250.3 576.7 577
*typical UV/MSD retention times **as PP derivative (as Isocyanate) in ng/µL

10.0 References
10.1 Draft Method 207
10.2 Conditional Test Method 024

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 Issued: 4/15/00
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Figure 1 Liquid chromatograms of a 10.0-ng/µL solution of Isocyanate-PP Ureas.

DAD1 A, Sig=254,4 Ref=490,40 (SNAPSHOT.D)

DAD1 B, Sig=240,4 Ref=490,40 (SNAPSHOT.D)
MSD1 TIC, MS File (SNAPSHOT.D) APCI, Pos, SIM
MSD1 501, EIC=500.9:501.9 (SNAPSHOT.D) APCI, Pos, SIM HDI,TDI,MDI-PP
MSD1 577, EIC=576.9:577.9 (SNAPSHOT.D) APCI, Pos, SIM
MSD1 495, EIC=494.9:495.9 (SNAPSHOT.D) APCI, Pos, SIM
254 nm, 240 nm and SIM
Norm.

1.727
6.693
7.044
700000

600000

500000

1.846
400000

6.239

5.907
300000

2.014
3.099
3.670
200000
.8
26

2.526
1

2.851
6.692

: 11
ea

3.417
100000 Ar
5.908

1.497
3.098

2.521
3.669

2.843
4.525 4.523

1.401
1.867
10.945
11.640
11.671

0.449
0

0 2 4 6 8 10 min

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 Issued: 4/15/00
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DAD1 A, Sig=254,4 Ref=490,40 (SNAPSHOT.D)

Norm.

6.693
300 UV @
4,4 MDI-pp
250

200
DMSO

150 2,4 MDI-pp

1.846
100
HDI-pp

5.907
2,6 TDI-pp
50

2.014
3.099
3.670
2,4 TDI-pp

2.526
2.851
0

4.523

1.497
-50

0 2 4 6 8 10 min

DAD1 B, Sig=240,4 Ref=490,40 (SNAPSHOT.D)

Norm.
UV @ 240

1.727
1400

1200
DMSO

1000

800

600

400
2,6 TDI-pp
4,4 MDI-pp
HDI-pp
2,4 MDI-pp
6.692

200
2,4 TDI-pp
5.908

3.098

2.521
3.669

2.843
0
4.525

1.401
0 2 4 6 8 10 min

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 Issued: 4/15/00
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MSD1 TIC, MS File (10020\10020003.D) APCI, Pos, SIM, Frag: 150

Norm.
MSD

7.044
700000
4,4 MDI-

600000
2,4 MDI-
500000

400000
HDI-

6.239
300000
2,6 TDI- 06
+0
28e
200000 2,4 TDI-
29
.3 6
:1 76
ea 54
r 8

3.440
100000 A :
ea

4.021
Ar

1.867
13.590

0.449
10.945
11.650
14.529
15.049

2 4 6 8 10 12 14 min

495.2 - HDI-
501.2 - 2,6 TDI- 501.2 - 2,4 TDI- 577.2 - 2,4 MDI- 577.2 - 4,4 MDI-
*MSD1 SPC, time=3.423 of 10020\10020 *MSD1 SPC, time=4.000 of 10020\100200 *MSD1 SPC, time=6.215 of 10020\1 *MSD1 SPC, time=7.296 of 10020\1002

100 100 100 100

501.2
501.2
577.2
577.2

Max: Max: 4 Max: Max: 3

495.2
80 80 80 80

60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0

500 520 540 560 m 500 520 540 560 m 500 520 540 560 m 500 520 540 560 m

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MSD1 495, EIC=494.9:495.9 (10020\10020003.D) APCI, Pos, SIM, Frag: 150

Norm.
MSD EIC @ 495.2
35000
HDI-pp
30000
HDI-pp
25000

20000

15000

10000

8 55
5000 74
5
e a:

2.937
Ar
0

2 4 6 8 10 12 14 min
MSD1 501, EIC=500.9:501.9 (10020\10020003.D) APCI, Pos, SIM, Frag: 150
Norm.

MSD EIC @ 501.2

40000 TDI-pp

2,6 TDI-pp 2,4 TDI-pp

30000

20000

13
39
10000 : 73
5 ea

3.808
08 Ar
15
:7
ea

2.987
Ar
0

2 4 6 8 10 12 14 min
MSD1 577, EIC=576.9:577.9 (10020\10020003.D) APCI, Pos, SIM, Frag: 150
Norm.

700000 MSD EIC @ 577.2

600000
MDI-pp
500000 4,4’ MDI-pp
400000

300000
2,4’ MDI-pp 7
00
200000 6 e+
00 342
e+ . 40
386 :2
100000 30 ea
6.624

: 7. Ar
ea
5.571

0 Ar

2 4 6 8 10 12 14 min

Isocyanates-11