Biomarkers in Hydrolysed Urine, Plasma and Erythrocytes Among Workers Exposed to Thermal Degradation Products From Toluene Diisocyanate Foam Pernilla Lind, Marianne Dalene, Håkan Tinnerberg and Gunnar Skarping* Department of Occupational and Environmental Medicine, University Hospital, S-221 85 Lund, Sweden Blood and urine samples were collected from six workers and two volunteers exposed to thermal degradation products from toluene diisocyanate (TDI)-based polyurethane (PUR) before and during the summer vacation.Air samples were collected on filters impregnated with 9-(N-methylaminomethyl)anthracene. The concentrations of the amines corresponding to 2,4- and 2,6-TDI, i.e., 2,4- and 2,6-toluenediamine (TDA), were determined in urine (U-TDA), plasma (P-TDA) and erythrocytes (E-TDA) after acid hydrolysis as pentafluoropropionic anhydride derivatives by GC–MS. Among the workers urinary elimination phases were seen. The estimated medians of the urinary half-lives were for the slow phase 18 d for 2,4-TDA and 19 d for 2,6-TDA.P-2,4-TDA ranged between 2.5 and 19 ng ml21 and P-2,6-TDA between 4.4 and 30 ng ml21. The estimated median of the half-lives in plasma were 7.8 d for 2,4-TDA and 9.6 d for 2,6-TDA. E-2,4-TDA ranged between 0.5 and 6.6 ng g21 and E-2,6-TDA between 1.2 and 14 ng g21. A significant linear relationship was found between the mean P-TDA and the mean E-TDA. Linear relationships were observed between the mean daily U-TDA and P-TDA and E-TDA.Virtually linear relationships were obtained for P-TDA and E-TDA and the TDI air levels. Proteins from lysed erythrocytes were separated and fractionated by gel filtration. ‘TDI’-modified proteins were found in six out of a total of 80 fractions (fractions 51–56). These co-eluted completely with the haemoglobin (UV, 415 nm). Fractions 51–56 contained 89% of the applied amounts of 2,4-TDA and 81% of 2,6-TDA. Keywords: Biomarkers; gas chromatography–mass spectrometry; toluene diisocyanate; toluenediamine; thermal degradation Occupational exposure to isocyanates is associated with respiratory disease.1–3 The main applications of isocyanates are in the manufacture of elastomers, rigid and flexible polyurethane (PUR) foams, glues and lacquers.Toluene diisocyanate (TDI) dominates in the production of flexible foam and the 2,4- and 2,6-isomers are used typically mixed in a ratio of 80 : 20. Exposure to isocyanates may occur when PUR is thermally degraded.The presence of high concentrations of complex isocyanates, aminoisocyanates and amines has recently been observed in the work atmosphere.4 The methods available for the determination of biomarkers are based on the determination of the amines corresponding to the isocyanates in hydrolysed biological fluids, e.g., 2,4- and 2,6-toluenediamine (TDA).5,6 A detection limit of 0.05 mg l21 of TDA and a precision of 3.5% for 2,4-TDA and 1.6% for 2,6-TDA were found.The amines are liberated under strongly acidic or alkaline hydrolysis conditions, indicating the presence of adducts to proteins and peptides.7 Adducts to plasma albumin among workers exposed to TDI8 and haemoglobin among workers exposed to methylenediphenyl diisocyanate and methylenedianiline have been demonstrated.9 In a study of workers occupationally exposed to TDI, the concentrations of TDA in plasma showed only limited variation during work-days while the concentrations in urine varied considerably.10 The estimated mean half-lives in plasma were 21 (range 14–34) d for 2,4-TDA and 21 (16–26) d for 2,6-TDA, and the urinary half-lives were 5.8–11 d for 2.4-TDA and 6.4–9.3 d for 2,6-TDA.11 Volunteers exposed to TDI showed urinary half-lives of about 1.9 h for 2,4-TDA and 1.6 h for 2,6-TDA in a first phase and of 5 h in a second phase12 and the half-lives in plasma from volunteers were 2–5 h of 2,4- and 2,6-TDA in a first phase and > 6 d in a second phase.13 The aim of this study was to determine the concentrations of TDA in hydrolysed urine (U-TDA), plasma (P-TDA) and erythrocytes (E-TDA) and their relationships in workers exposed to thermal degradation products of TDI-PUR before and during an exposure-free period.A knowledge of the relationships and variations in time between U-TDA, P-TDA and E-TDA is necessary for the sampling strategy and exposure control. Experimental Apparatus The hydrolysed biological samples were analysed with an MD 800 quadrupole mass spectrometer (Fisons Instruments, Altrincham, UK) connected to a GC 8000 gas chromatograph equipped with an A200S autosampler (Fisons Instruments, Milan, Italy).The 2,4- and 2,6-TDA-pentafluoropropionic anhydride (PFPA) derivatives were determined by monitoring the ions at m/z 394 corresponding to the M 2 20 ions and m/z 397 corresponding to the M 2 20 ions of the trideuteriumlabelled internal standards (M = molecular mass).A fusedsilica capillary column with a chemically bonded stationary phase, DB-5 (J & B Scientific, Folsom, CA, USA), 25 m 30.25 mm id with a film thickness of 0.25 mm, was used. Detailed chromatographic and mass spectrometric information was given previously.5 A Waters Model 600 multisolvent delivery system (Millipore –Waters, Milford, MA, USA), a Waters Model 712 WISP and a Waters Model 490 UV detector were used for the determination of isocyanates in air and for the gel separation of haemoglobin.Haemoglobin was fractionated using a Pharmacia –LKB SuperFrac (Pharmacia, Uppsala, Sweden). Chemicals Both 2,4- and 2,6-TDA were obtained from Fluka (Buchs, Switzerland), HCl, NaOH, H2SO4 and K2HPO4 from Merck (Darmstadt, Germany), pentafluoropropionic anhydride (PFPA) from Pierce (Rockford, IL, USA), toluene, isooctane and HPLC-grade acetonitrile from Lab-Scan (Dublin, Ireland), technical-grade TDI (80 + 20 2,4- + 2,6-TDI) and triethylamine (99%) from Janssen (Beerse, Belgium), NH4HCO3 from BDH Analyst, January 1997, Vol. 122 (51–56) 51(Poole, UK), 9-(N-methylaminomethyl)anthracene (MAMA) from Aldrich Chemie (Steinham, Germany), haemoglobin from Sigma (St. Louis, MO, USA) and trideuterated 2,4- and 2,6-TDA [CD3C6H3(NH2)2, TDDA], used as internal standards (IS) from Synthelec (Lund, Sweden). Subjects and Exposure Six workers (A–F) and two volunteers (colleagues) participated in this study. The work took place in a flame lamination factory applying a thin layer of TDI-based flexible foam on to textile fabric.The surface of the PUR foam was partly melted by an open flame immediately prior to the application. The work took place (7 am–4 pm) in two large workshops with an open door in between. Workers A, D, E and F were involved in the lamination process and workers B and C were occupied with cutting the produced laminated fabric. The lamination was performed in both workshops, but not at the same time.Worker D was employed only during the summer and was assumed not to be chronically exposed. The two volunteers were mainly involved in collecting air samples. Procedure Preparation of standard solutions TDI standard solutions were prepared by dissolving TDI in isooctane and further dilutions were made in toluene. Standard solutions of 2,4- and 2,6-TDA were prepared by dissolving accurately weighed amounts in 1 m HCl. The internal standards, 2,4- and 2,6-TDDA, were dissolved in 1 m H2SO4.Air sampling on impregnated glass-fibre filters Three subsequent air samples were collected in the breathing zone per worker during one typical work day of the study during about 8 h. The sampling times of each air sample varied between 100 and 220 min. The average exposure was calculated by taking the total amount of 2,4- and 2,6-TDI on the three filters divided by the total sampling time. Air samples were collected (1 l min21) on 13 mm glass-fibre filters, in a Teflon filter holder, impregnated with a MAMA–glycerol mixture.14 Immediately after sampling, the filters were placed in a test-tube containing 4 ml of toluene. The excess of the MAMA reagent was extracted for 5 min with 2 ml of 0.1 m HCl added to the testtube.A 2 ml volume of the toluene solution was separated and evaporated to dryness. The dry residue was dissolved in 1 ml of the mobile phase added before the LC–UV (254 nm) determinations. The LC column was a Hypersil BDS C18 (150 3 4.6 mm id) with 5 mm particles.The mobile phase (1.5 ml min21) consisted of acetonitrile–water (70 + 30) containing 3% triethylamine in the water and adjusted to pH 3 with phosphoric acid. Peak-height measurements were made and compared with a calibration plot for reagent solutions spiked with TDI to six concentrations in the range 0–1.2 mg ml21. Collection of biological samples Blood and urine samples were collected from six workers (A–F) before and during their summer vacation.Sampling was performed both at the factory and in the workers’ homes. All urine produced during 2 d of exposure and on three exposurefree days were collected. A few urine samples were also collected on different occasions during the 3–4 week vacation. Blood samples were taken once a day during 2 d of exposure and on three exposure-free days. Blood and urine samples were also taken from two volunteers. All urine produced during the day of exposure and the following exposure-free day was collected.Blood samples were taken on three occasions at the day of exposure and two and five times, respectively, during the following eight exposure-free days. Sampling, handling and storing of biological samples All urine samples were collected in polyethylene bottles. The creatinine concentration [determined at the Department of Clinical Chemistry, University Hospital, Lund, by use of Kodak Ektachem clinical chemistry slides (CREA) and Kodak Ektachem 700 XR-C analyser], pH and total volume of each urine sample were determined.Blood was sampled in heparinized tubes (Venoject). Plasma was separated within 8 h and transferred to new tubes. The erythrocytes were washed in 0.9% NaCl two or three times. The urine, plasma and erythrocyte samples were kept frozen at 220 °C until analysis. Gel filtration of haemoglobin Washed erythrocytes from worker A were repeatedly frozen and thawed four times in a double volume of Millipore-purified water (Milli-Q system, Millipore, Bedford, MA, USA).The cell debris was removed by centrifugation at 18 500 g for 15 min. The haemoglobin-containing supernatant was diluted five fold in 50 mm NH4HCO3 and filtered using a 0.2 mm hydrophilic membrane filter (Minisart RC 25; Sartorius, G�ottingen, Germany). A volume of 200 ml of the solution was separated on a Superose 12 (10/30) gel filtration column (Pharmacia Biotech, Uppsala, Sweden) and fraction into 80 fractions of 0.25 ml each.The mobile phase consisted of 50 mm NH4HCO3 at a flow rate of 0.5 ml min21. UV traces were recorded at 415 nm. A total of 50 fractionations were performed and fractions were collected in the same subsequent fraction vials. Hence each fraction vial contained 12.5 ml. The 80 fractions were then evaporated to dryness and the dry residues were dissolved in 1 ml aliquots of Millipore-purified water. Hydrolysis To 1 ml of plasma, 1 ml of urine, 0.5 g of erythrocytes in 0.5 ml of Millipore-purified water or 1 ml of concentrated gel filtration fractions, 100 ml of IS were added together with 1.5 ml of 3 m H2SO4 before hydrolysis at 100 °C for 16 h.In the sample batches the calibration samples were prepared in the range 0–30 ng ml21 TDA with 5.5 ng ml21 IS for plasma, 0–10 ng ml21 TDA with 2.0 ng ml21 IS and 0–60 ng ml21 TDA with 11 ng ml21 IS for urine, 0–10 ng ml21 TDA with 2.0 n ml21 IS for erythrocytes and 0–5 ng ml21 TDA with 1.0 ng ml21 IS for the gel filtration fractions.After hydrolysis the samples were worked-up as described below. Work-up procedure A 5 ml volume of saturated NaOH and 2 ml of toluene were added to the samples. The mixtures were shaken for about 10 min and then centrifuged at 1500 g for 10 min. The organic layers were transferred into new test-tubes and 20 ml of PFPA were added to each. The mixtures were immediately shaken vigorously. The excess of the reagent and acid formed was removed by extraction with 2 ml of 1 m phosphate buffer solution (pH 7.5).The toluene layers containing the amide derivatives and the internal standards were transferred into 1.5 ml autosampler vials with Teflon seals. When analysing the gel filtration samples, about 1 ml of the toluene solution was evaporated to dryness and diluted with 100 ml of toluene. The samples were then ready for injection into the GC–MS system. Statistics To determine if the correlation coefficients are significant, we calculated a t-value: t = ýrý (n 2 2)1 2/(1 2 r2)1 2 (r = correlation 52 Analyst, January 1997, Vol. 122coefficient, n = number of observations). The calculated value of t was compared with the tabulated value at the 95% significance level, using a two-tailed t-test and n 2 2 degrees of freedom. If the calculated value of t was greater than the tabulated value, a significant correlation exists. Results Isocyanates in Air The average air concentrations during the work day of 2,4- and 2,6-TDI at the breathing zone using personal sampling were for worker A 3.5 and 10 mg m23, B 0.9 and 2.6 mg m23, C 1.8 and 6.5 mg m23, D 1.4 and 4.7 mg m23, E 1.6 and 4.1 mg m23 and F 2.1 and 7.4 mg m23, respectively.No representative air samples for the volunteers were collected. Biomarkers in Hydrolysed Urine The U-TDA for workers A–F are shown in Fig. 1. During the work U-TDA was rapidly eliminated and a slower elimination phase were seen during the exposure-free period.For workers B and E one urine sample was considerably above the assumed ideal elimination curve. This was probably due to an unnoticed exposure to TDA dust at the factory after cessation of flame lamination work. The urinary half-lives in the slower phase were estimated by plotting the natural logarithm of the urinary concentrations against time and by dividing ln 2 with the calculated slopes. The half-lives of 2,4- and 2,6-TDA were estimated for worker A to be 32 (r = 0.82, n = 10) and 25 d (r = 0.94, n = 10), B 20 (r = 0.67, n = 23) and 20 d (r = 0.90, n = 23), C 15 (r = 0.93, n = 9) and 18 d (r = 0.91, n = 9), D 4.3 (r = 0.90, n = 8) and 11 d (r = 0.79, n = 8), E 26 (r = 0.36, n = 23) and 22 d (r = 0.88, n = 24) and F 14 (r = 0.93, n = 12) and 16 d (r = 0.97, n = 12), respectively.The medians of the urinary half-lives were for the slow phase 18 d for 2,4-TDA and 19 d for 2,6-TDA. The urinary half-lives for the two volunteers were calculated to 5.3 (r = 0.96, n = 4) and 6.2 h (r = 0.97, n = 4) h for 2,4-TDA and 8.4 (r = 0.96, n = 5) and 7.4 h (r = 0.99, n = 4) for 2,6-TDA (Fig. 2). Biomarkers in Plasma and Erythrocytes The concentrations of 2,4-TDA in plasma were in the range 2.5–19 ng ml21 and those of 2,6-TDA in the range 4.4–30 ng ml21 among the workers. In erythrocytes, the concentrations of 2,4-TDA were in the range 0.5–6.6 ng g21 and those of 2,6-TDA in the range 1.2–14 ng g21 among the workers.Significant linear relationships were seen between the mean concentrations, for all individual samples, in plasma and erythrocytes of 2,4-TDA (r = 0.97, n = 6) and 2,6-TDA (r = 0.97, n = 6) (Fig. 3). P-TDA declined slowly after work cessation. The half-lives in plasma (calculated as for UTDA) were in the range 6.5–12 d for 2,4-TDA (r = 0.76–0.95, n = 4) and 7–11 d for 2,6-TDA (r = 0.76–0.97, n = 4). E-TDA declined too little for the calculation of half-lives.When plotting the mean daily urinary concentrations adjusted for creatinine against P-TDA and E-TDA, relationships were observed. The relationships became more linear with the number of days after cessation of work (Fig. 4). Significant correlations were seen, with the exception of E-2,4-TDA on day 2. Significant linear plots, with the exception of E-2,6-TDA, were obtained on plotting the P-TDA and E-TDA for workers A, B, C, E and F against the TDI air levels measured on day 1 (worker D was only employed during the summer) (Fig. 5). No detectable P-TDA or E-TDA were found in the two volunteers, except for very low concentrations of 2,4-TDA ( < 0.33 ng ml21) in plasma from one of the volunteers. Among a group of 17 unexposed humans, the-TDA and P-TDA were below the detection limit. Conditions for the Hydrolysis of Haemoglobin Volumes of 1 ml of the haemoglobin-containing supernatant (see Gel filtration of haemoglobin) were hydrolysed in 1.5 ml of 3 m H2SO4 at 100 °C for 4, 8, 16, 24 and 48 h.After hydrolysis the samples were subjected to the work-up procedure. No free 2,4- and 2,6-TDA were seen without hydrolysis. The release of 2,4- and 2,6-TDA increased with increasing hydrolysis time (Fig. 6). For the preparation of haemoglobin from the erythrocytes, the recoveries of 2,4- and 2,6-TDA were 77% and 91%, respectively. On analysing the remaining cell debris in the centrifuge tube no TDA was found. Gel Filtration of Proteins in erythrocytes TDA were only found in six of the 80 fractions (fractions 51–56) from the gel filtration separation and co-eluted com- Fig. 1 Concentrations of 2,4-TDA (2) and 2,6-TDA (5) in hydrolysed urine (mg per mmol of creatinine) from workers A–F.All urine produced during 2 work days and on the three days following work cessation (vacation), and on different occasions during the following 3–4 weeks, was collected. Time 0 = 6.00 am on the first day of the study.Analyst, January 1997, Vol. 122 53pletely with the haemoglobin (UV, 415 nm) peak. Fractions 51–56 contained 89% of the applied amounts of 2,4-TDA and 81% of 2,6-TDA (Fig. 7). In the GC–MS traces pure and well separated 2,4- and 2,6-TDA-PFPA peaks were seen. The 2,4-and 2,6-TDA-PFPA peaks in the chromatogram of fraction 54 were found at an unfiltered S/N of 175 and 61, respectively (rms). Only traces of TDA-PFPA were seen in fractions 1–50 and 57–80, due to impurities of TDA in the internal standards.These were also present in the chemical blanks. Discussion Isocyanates occur in many chemical and physical forms in workplace atmospheres. In the TDI foam industry, the monomers dominate in the gas phase. However, when particles are formed, many other compounds may be present in addition to monomers. These can be measured by methods which are based on the reaction of isocyanates with secondary amines containing a chromophore.14 During the thermal decomposition of PUR, in addition to isocyanates, aminoisocyanates and amines are also present.This means that isocyanate exposure measured by ‘conventional’ methods, underestimates the true exposure. It was therefore surprising, but encouraging, to find a relationship between the air data and the P-TDA and E-TDA levels among Fig. 2 Concentrations of 2,4-TDA (2) and 2,6-TDA (5) in hydrolysed urine (mg per mmol of creatinine) from two volunteers. The volunteers were present in the factory from 7 am until 4 pm on day 1.Time 0 = 6.00 am the first day of the study. Fig. 4 Concentrations of 2,4-TDA (5/-) and 2,6-TDA (2/8) in hydrolysed plasma (ng ml21) and erythrocytes (ng g21) and mean urinary concentration (mg per mmol of creatinine) during 1 d of exposure (day 2) and the three following exposure-free days (days 3–5) among six workers (A–F). Day 2: r P- 2,4-TDA = 0.84, r E-2,4-TDA = 0.81, r P-2,6-TDA = 0.86, r E-2,6-TDA = 0.83; Day 3: r P-2,4-TDA = 0.91, r E-2,4-TDA = 0.91, r P-2,6-TDA = 0.94, r E-2,6-TDA = 0.89; Day 4: r P-2,4-TDA = 0.92, r E-2,4-TDA = 0.98, r P-2,6-TDA = 0.97, r E-2,6-TDA = 0.98; Day 5: r P-2,4-TDA = 0.98, r E- 2,4-TDA = 0.97, r P-2,6-TDA = 0.99, r E-2,6-TDA = 0.97. Continuous line = 2,4-TDA; broken line = 2,6-TDA.Fig. 3 Mean concentrations, for all individual samples, of 2,4-TDA (5) and 2,6-TDA (-) in hydrolysed plasma (ng ml21) and erythrocytes (ng g21) among six workers (A–F) during 5 d. Continuous line = 2,4-TDA, r = 0.97; broken line = 2,6-TDA, r = 0.97. 54 Analyst, January 1997, Vol. 122the workers. However, the relationship was different to that for data from a flexible foam factory,11 where about the same levels in plasma were found but three times higher TDI air levels. This can be explained by the insufficiency and the limitations of the air methods used to measure more complex isocyanates. Data from conventional air methods may therefore be used as a relative index of exposure in the absence of methods that take into account all other isocyanates that may influence the industrial hygiene. Further studies of these aspects are in progress.The urine data clearly demonstrate fast elimination phases and one slower phase. It can be assumed that the fast phases reflect the more recent exposure and the slow phase reflects urinary elimination of degradation products of modified proteins, and hence the history of exposure. The urinary elimination half-lives for the slow phase were about the same as the half-life of albumin and the mean half-lives in plasma among TDI foam workers.11 The significant linear relationships between the U-TDA and E-TDA and P-TDA that improved with time after work cessation may also add to the explanation of the presence of modified erythrocytes and albumin metabolites and breakdown products excreted in urine.These products seem to dominate after a few days of work cessation. Worker D, a worker not chronically exposed, had shorter urinary elimination half-lives compared with the chronically exposed workers.The volunteers in this study had about the same urinary halflives as the volunteers in the volunteer study.12 The volunteer study may reflect an interesting difference in uptake, metabolism and excretion between chronically exposed workers and volunteers and the difference in exposure situations. In the workplace studies much more complex isocyanates and different chemical and physical forms are present in the air.Biomarkers of both 2,4- and 2,6-TDA were found in plasma and erythrocytes from all workers. The half-lives in plasma, in Fig. 5 Concentrations of 2,4-TDA (5) and 2,6-TDA (-) among workers A, B, C, E and F and in worker D (2 = 2,4-TDA) and (8 = 2,6-TDA) in: (a), hydrolysed plasma (ng ml21); and (b), hydrolysed erythrocytes (ng g21), plotted against the mean individual air concentrations of 2,4- and 2,6-TDI (mg m23) for the first work day of the study.For the calculation of the linear curves, data from worker D were excluded as he was a seasonal worker and not chronically exposed (r P-2,4-TDA = 0.98, r P- 2,6-TDA = 0.97, r E-2,4-TDA = 0.92, r E-2,6-TDA = 0.85). Fig. 7 Amounts of 2,4-TDA (5) and 2,6-TDA (~) (ng) in the fractions eluted from a gel filtration separation of lysed erythrocytes (cell debris separated) from worker A. Fractions 51–56 contained 89% of the applied amount of 2,4-TDA and 81% of 2,6-TDA and co-eluted with haemoglobin (UV 415 nm, broken line, 100% was saturated detection).Selected ion monitoring chromatogram of fraction 43 represents a fraction not containing TDA, and that of fraction 54 one with ‘TDI’-modified compounds. (m/z = 394.2 is the M 2 20 ions of TDA-PFPA and m/z = 397.2 is the M 2 20 ions of TDDA-PFPA). Fig. 6 Concentrations of 2,4-TDA (5) and 2,6-TDA (-) in lysed erythrocytes (cell debris separated) after hydrolysis for 0, 4, 8, 16, 24 and 48 h at 100 °C.Analyst, January 1997, Vol. 122 55this study, were about 50% shorter than those in the flexible foam worker study11 but about the same as those in the volunteer study.13 The difference may be explained by the presence of a faster phase, but this could not be observed in the study of flexible foam workers as blood samplings were performed at too long intervals. The E-TDA levels were virtually stable during the study and therefore no half-lives could be calculated.They can therefore be assumed to be longer than for plasma. A significant linear relationship between PTDA and E-TDA was observed. No TDA was found in the centrifuged and lysed erythrocyte solution on performing the work-up procedure without hydrolysis. This indicates covalently TDI-modified proteins. The gel filtration demonstrated that the modified protein in fact was haemoglobin. The hydrolysis pattern of haemoglobin was found to be almost the same as for plasma among TDI-exposed workers.7 This is an indication that the nature of the chemical bondings is the same and that the same hydrolysis conditions can be used.There are many aspects of the uptake, metabolism and excretion of isocyanates that are not yet fully understood. However, there are many reasons to believe that modified proteins and peptides are involved in isocyanate-associated disease. It is therefore interesting to know that intracellular in addition to intercellular proteins are modified with TDI among workers.The mechanism by which isocyanates are able to pass cell membranes is not known and further studies on this aspect are in progress. The present study has shown that the history of exposure can be determined by biomarkers in plasma and erythrocytes. Recent exposure does not greatly affect the P-TDA and E-TDA levels. Biomarkers in urine can be used for the same purpose if sampling is performed after the fast elimination phases. This work was supported by the Swedish Work Environment Fund.We thank M. Adamsson, M. Spanne and T. Russin for skilful technical assistance and Professor A. Grubb and Associate Professor J.-O. Jeppson for valuable discussions. References 1 Banks, D. E., Butcher, B. T., and Salviaggio, J. E., Ann. Allergy, 1986, 57, 389. 2 Baur, X., Marek, W., Ammon, J., Czuppon, A. B., Marczynski, B., Raulf-Heimsoth, M., Roemmelt, H., and Fruhman, G., Int. Arch. Occup. Environ. Health, 1994, 7, 310. 3 Vandenplas, O., Malo, J. L., Saetta, M., Mapp, C. E., and Fabbri, L. M., Br. J. Ind. Med., 1993, 50, 213. 4 Tinnerberg, H., Spanne, M., Dalene, M., and Skarping, G., Analyst, 1996, 121, 1101. 5 Skarping, G., Dalene, M., and Lind, P., J. Chromatogr., 1994, 663, 199. 6 Maitre, A., Berode, M., Perdrix, A., Romazini, S., and Savolainen, H., Int. Arch. Occup. Environ. Health, 1993, 65, 97. 7 Lind, P., Skarping, G., and Dalene, M., Anal. Chim. Acta, in the press. 8 Lind, P., Dalene, M., Lindstr�om, V., Grubb, A., and Skarping, G., Analyst, 1997, 122, in the press. 9 Sch�utze, D., Sepai, O., Lewalter, J., Miksche, L., Henschler, D., and Sabbioni, G., Carcinogenesis, 1995, 16, 573. 10 Persson, P., Dalene, M., Skarping, G., Adamsson, M., and Hagmar, L., Br. J. Ind. Med., 1993, 50, 1111. 11 Lind, P., Dalene, M., Skarping, G., and Hagmar, L., Occup. Environ. Med., 1996, 53, 94. 12 Skarping, G., Brorson, T., and Sang�o, C., Int. Arch. Occup. Environ. Health, 1991, 63, 83. 13 Brorson, T., Skarping, G., and Sang�o, C., Int. Arch. Occup. Environ. Health, 1991, 63, 253. 14 Tinnerberg, H., Dalene, M., and Skarping, G., Am. Ind. Hyg. Assoc. J., in the press. Paper 6/06148F Received September 6, 1996 Accepted November 6, 1996 56 Analyst, January 1997, Vol. 122 Biomarkers in Hydrolysed Urine, Plasma and Erythrocytes Among Workers Exposed to Thermal Degradation Products From Toluene Diisocyanate Foam Pernilla Lind, Marianne Dalene, Håkan Tinnerberg and Gunnar Skarping* Department of Occupational and Environmental Medicine, University Hospital, S-221 85 Lund, Sweden Blood and urine samples were collected from six workers and two volunteers exposed to thermal degradation products from toluene diisocyanate (TDI)-based polyurethane (PUR) before and during the summer vacation.Air samples were collected on filters impregnated with 9-(N-methylaminomethyl)anthracene. The concentrations of the amines corresponding to 2,4- and 2,6-TDI, i.e., 2,4- and 2,6-toluenediamine (TDA), were determined in urine (U-TDA), plasma (P-TDA) and erythrocytes (E-TDA) after acid hydrolysis as pentafluoropropionic anhydride derivatives by GC–MS.Among the workers urinary elimination phases were seen. The estimated medians of the urinary half-lives were for the slow phase 18 d for 2,4-TDA and 19 d for 2,6-TDA. P-2,4-TDA ranged between 2.5 and 19 ng ml21 and P-2,6-TDA between 4.4 and 30 ng ml21. The estimated median of the half-lives in plasma were 7.8 d for 2,4-TDA and 9.6 d for 2,6-TDA.E-2,4-TDA ranged between 0.5 and 6.6 ng g21 and E-2,6-TDA between 1.2 and 14 ng g21. A significant linear relationship was found between the mean P-TDA and the mean E-TDA. Linear relationships were observed between the mean daily U-TDA and P-TDA and E-TDA. Virtually linear relationships were obtained for P-TDA and E-TDA and the TDI air levels. Proteins from lysed erythrocytes were separated and fractionated by gel filtration.‘TDI’-modified proteins were found in six out of a total of 80 fractions (fractions 51–56). These co-eluted completely with the haemoglobin (UV, 415 nm). Fractions 51–56 contained 89% of the applied amounts of 2,4-TDA and 81% of 2,6-TDA. Keywords: Biomarkers; gas chromatography–mass spectrometry; toluene diisocyanate; toluenediamine; thermal degradation Occupational exposure to isocyanates is associated with respiratory disease.1–3 The main applications of isocyanates are in the manufacture of elastomers, rigid and flexible polyurethane (PUR) foams, glues and lacquers.Toluene diisocyanate (TDI) dominates in the production of flexible foam and the 2,4- and 2,6-isomers are used typically mixed in a ratio of 80 : 20. Exposure to isocyanates may occur when PUR is thermally degraded. The presence of high concentrations of complex isocyanates, aminoisocyanates and amines has recently been observed in the work atmosphere.4 The methods available for the determination of biomarkers are based on the determination of the amines corresponding to the isocyanates in hydrolysed biological fluids, e.g., 2,4- and 2,6-toluenediamine (TDA).5,6 A detection limit of 0.05 mg l21 of TDA and a precision of 3.5% for 2,4-TDA and 1.6% for 2,6-TDA were found.The amines are liberated under strongly acidic or alkaline hydrolysis conditions, indicating the presence of adducts to proteins and peptides.7 Adducts to plasma albumin among workers exposed to TDI8 and haemoglobin among workers exposed to methylenediphenyl diisocyanate and methylenedianiline have been demonstrated.9 In a study of workers occupationally exposed to TDI, the concentrations of TDA in plasma showed only limited variation during work-days while the concentrations in urine varied considerably.10 The estimated mean half-lives in plasma were 21 (range 14–34) d for 2,4-TDA and 21 (16–26) d for 2,6-TDA, and the urinary half-lives were 5.8–11 d for 2.4-TDA and 6.4–9.3 d for 2,6-TDA.11 Volunteers exposed to TDI showed urinary half-lives of about 1.9 h for 2,4-TDA and 1.6 h for 2,6-TDA in a first phase and of 5 h in a second phase12 and the half-lives in plasma from volunteers were 2–5 h of 2,4- and 2,6-TDA in a first phase and > 6 d in a second phase.13 The aim of this study was to determine the concentrations of TDA in hydrolysed urine (U-TDA), plasma (P-TDA) and erythrocytes (E-TDA) and their relationships in workers exposed to thermal degradation products of TDI-PUR before and during an exposure-free period.A knowledge of the relationships and variations in time between U-TDA, P-TDA and E-TDA is necessary for the sampling strategy and exposure control. Experimental Apparatus The hydrolysed biological samples were analysed with an MD 800 quadrupole mass spectrometer (Fisons Instruments, Altrincham, UK) connected to a GC 8000 gas chromatograph equipped with an A200S autosampler (Fisons Instruments, Milan, Italy). The 2,4- and 2,6-TDA-pentafluoropropionic anhydride (PFPA) derivatives were determined by monitoring the ions at m/z 394 corresponding to the M 2 20 ions and m/z 397 corresponding to the M 2 20 ions of the trideuteriumlabelled internal standards (M = molecular mass).A fusedsilica capillary column with a chemically bonded stationary phase, DB-5 (J & B Scientific, Folsom, CA, USA), 25 m 30.25 mm id with a film thickness of 0.25 mm, was used.Detailed chromatographic and mass spectrometric information was given previously.5 A Waters Model 600 multisolvent delivery system (Millipore –Waters, Milford, MA, USA), a Waters Model 712 WISP and a Waters Model 490 UV detector were used for the determination of isocyanates in air and for the gel separation of haemoglobin. Haemoglobin was fractionated using a Pharmacia –LKB SuperFrac (Pharmacia, Uppsala, Sweden). Chemicals Both 2,4- and 2,6-TDA were obtained from Fluka (Buchs, Switzerland), HCl, NaOH, H2SO4 and K2HPO4 from Merck (Darmstadt, Germany), pentafluoropropionic anhydride (PFPA)erce (Rockford, IL, USA), toluene, isooctane and HPLC-grade acetonitrile from Lab-Scan (Dublin, Ireland), technical-grade TDI (80 + 20 2,4- + 2,6-TDI) and triethylamine (99%) from Janssen (Beerse, Belgium), NH4HCO3 from BDH Analyst, January 1997, Vol. 122 (51–56) 51(Poole, UK), 9-(N-methylaminomethyl)anthracene (MAMA) from Aldrich Chemie (Steinham, Germany), haemoglobin from Sigma (St.Louis, MO, USA) and trideuterated 2,4- and 2,6-TDA [CD3C6H3(NH2)2, TDDA], used as internal standards (IS) from Synthelec (Lund, Sweden). Subjects and Exposure Six workers (A–F) and two volunteers (colleagues) participated in this study. The work took place in a flame lamination factory applying a thin layer of TDI-based flexible foam on to textile fabric. The surface of the PUR foam was partly melted by an open flame immediately prior to the application.The work took place (7 am–4 pm) in two large workshops with an open door in between. Workers A, D, E and F were involved in the lamination process and workers B and C were occupied with cutting the produced laminated fabric. The lamination was performed in both workshops, but not at the same time. Worker D was employed only during the summer and was assumed not to be chronically exposed. The two volunteers were mainly involved in collecting air samples.Procedure Preparation of standard solutions TDI standard solutions were prepared by dissolving TDI in isooctane and further dilutions were made in toluene. Standard solutions of 2,4- and 2,6-TDA were prepared by dissolving accurately weighed amounts in 1 m HCl. The internal standards, 2,4- and 2,6-TDDA, were dissolved in 1 m H2SO4. Air sampling on impregnated glass-fibre filters Three subsequent air samples were collected in the breathing zone per worker during one typical work day of the study during about 8 h. The sampling times of each air sample varied between 100 and 220 min.The average exposure was calculated by taking the total amount of 2,4- and 2,6-TDI on the three filters divided by the total sampling time. Air samples were collected (1 l min21) on 13 mm glass-fibre filters, in a Teflon filter holder, impregnated with a MAMA–glycerol mixture.14 Immediately after sampling, the filters were placed in a test-tube containing 4 ml of toluene.The excess of the MAMA reagent was extracted for 5 min with 2 ml of 0.1 m HCl added to the testtube. A 2 ml volume of the toluene solution was separated and evaporated to dryness. The dry residue was dissolved in 1 ml of the mobile phase added before the LC–UV (254 nm) determinations. The LC column was a Hypersil BDS C18 (150 3 4.6 mm id) with 5 mm particles. The mobile phase (1.5 ml min21) consisted of acetonitrile–water (70 + 30) containing 3% triethylamine in the water and adjusted to pH 3 with phosphoric acid.Peak-height measurements were made and compared with a calibration plot for reagent solutions spiked with TDI to six concentrations in the range 0–1.2 mg ml21. Collection of biological samples Blood and urine samples were collected from six workers (A–F) before and during their summer vacation. Sampling was performed both at the factory and in the workers’ homes. All urine produced during 2 d of exposure and on three exposurefree days were collected.A few urine samples were also collected on different occasions during the 3–4 week vacation. Blood samples were taken once a day during 2 d of exposure and on three exposure-free days. Blood and urine samples were also taken from two volunteers. All urine produced during the day of exposure and the following exposure-free day was collected. Blood samples were taken on three occasions at the day of exposure and two and five times, respectively, during the following eight exposure-free days.Sampling, handling and storing of biological samples All urine samples were collected in polyethylene bottles. The creatinine concentration [determined at the Department of Clinical Chemistry, University Hospital, Lund, by use of Kodak Ektachem clinical chemistry slides (CREA) and Kodak Ektachem 700 XR-C analyser], pH and total volume of each urine sample were determined. Blood was sampled in heparinized tubes (Venoject). Plasma was separated within 8 h and transferred to new tubes.The erythrocytes were washed in 0.9% NaCl two or three times. The urine, plasma and erythrocyte samples were kept frozen at 220 °C until analysis. Gel filtration of haemoglobin Washed erythrocytes from worker A were repeatedly frozen and thawed four times in a double volume of Millipore-purified water (Milli-Q system, Millipore, Bedford, MA, USA). The cell debris was removed by centrifugation at 18 500 g for 15 min. The haemoglobin-containing supernatant was diluted five fold in 50 mm NH4HCO3 and filtered using a 0.2 mm hydrophilic membrane filter (Minisart RC 25; Sartorius, G�ottingen, Germany).A volume of 200 ml of the solution was separated on a Superose 12 (10/30) gel filtration column (Pharmacia Biotech, Uppsala, Sweden) and fraction into 80 fractions of 0.25 ml each. The mobile phase consisted of 50 mm NH4HCO3 at a flow rate of 0.5 ml min21. UV traces were recorded at 415 nm. A total of 50 fractionations were performed and fractions were collected in the same subsequent fraction vials.Hence each fraction vial contained 12.5 ml. The 80 fractions were then evaporated to dryness and the dry residues were dissolved in 1 ml aliquots of Millipore-purified water. Hydrolysis To 1 ml of plasma, 1 ml of urine, 0.5 g of erythrocytes in 0.5 ml of Millipore-purified water or 1 ml of concentrated gel filtration fractions, 100 ml of IS were added together with 1.5 ml of 3 m H2SO4 before hydrolysis at 100 °C for 16 h.In the sample batches the calibration samples were prepared in the range 0–30 ng ml21 TDA with 5.5 ng ml21 IS for plasma, 0–10 ng ml21 TDA with 2.0 ng ml21 IS and 0–60 ng ml21 TDA with 11 ng ml21 IS for urine, 0–10 ng ml21 TDA with 2.0 n ml21 IS for erythrocytes and 0–5 ng ml21 TDA with 1.0 ng ml21 IS for the gel filtration fractions. After hydrolysis the samples were worked-up as described below. Work-up procedure A 5 ml volume of saturated NaOH and 2 ml of toluene were added to the samples.The mixtures were shaken for about 10 min and then centrifuged at 1500 g for 10 min. The organic layers were transferred into new test-tubes and 20 ml of PFPA were added to each. The mixtures were immediately shaken vigorously. The excess of the reagent and acid formed was removed by extraction with 2 ml of 1 m phosphate buffer solution (pH 7.5). The toluene layers containing the amide derivatives and the internal standards were transferred into 1.5 ml autosampler vials with Teflon seals.When analysing the gel filtration samples, about 1 ml of the toluene solution was evaporated to dryness and diluted with 100 ml of toluene. The samples were then ready for injection into the GC–MS system. Statistics To determine if the correlation coefficients are significant, we calculated a t-value: t = ýrý (n 2 2)1 2/(1 2 r2)1 2 (r = correlation 52 Analyst, January 1997, Vol. 122coefficient, n = number of observations).The calculated value of t was compared with the tabulated value at the 95% significance level, using a two-tailed t-test and n 2 2 degrees of freedom. If the calculated value of t was greater than the tabulated value, a significant correlation exists. Results Isocyanates in Air The average air concentrations during the work day of 2,4- and 2,6-TDI at the breathing zone using personal sampling were for worker A 3.5 and 10 mg m23, B 0.9 and 2.6 mg m23, C 1.8 and 6.5 mg m23, D 1.4 and 4.7 mg m23, E 1.6 and 4.1 mg m23 and F 2.1 and 7.4 mg m23, respectively.No representative air samples for the volunteers were collected. Biomarkers in Hydrolysed Urine The U-TDA for workers A–F are shown in Fig. 1. During the work U-TDA was rapidly eliminated and a slower elimination phase were seen during the exposure-free period. For workers B and E one urine sample was considerably above the assumed ideal elimination curve. This was probably due to an unnoticed exposure to TDA dust at the factory after cestion of flame lamination work.The urinary half-lives in the slower phase were estimated by plotting the natural logarithm of the urinary concentrations against time and by dividing ln 2 with the calculated slopes. The half-lives of 2,4- and 2,6-TDA were estimated for worker A to be 32 (r = 0.82, n = 10) and 25 d (r = 0.94, n = 10), B 20 (r = 0.67, n = 23) and 20 d (r = 0.90, n = 23), C 15 (r = 0.93, n = 9) and 18 d (r = 0.91, n = 9), D 4.3 (r = 0.90, n = 8) and 11 d (r = 0.79, n = 8), E 26 (r = 0.36, n = 23) and 22 d (r = 0.88, n = 24) and F 14 (r = 0.93, n = 12) and 16 d (r = 0.97, n = 12), respectively.The medians of the urinary half-lives were for the slow phase 18 d for 2,4-TDA and 19 d for 2,6-TDA. The urinary half-lives for the two volunteers were calculated to 5.3 (r = 0.96, n = 4) and 6.2 h (r = 0.97, n = 4) h for 2,4-TDA and 8.4 (r = 0.96, n = 5) and 7.4 h (r = 0.99, n = 4) for 2,6-TDA (Fig. 2). Biomarkers in Plasma and Erythrocytes The concentrations of 2,4-TDA in plasma were in the range 2.5–19 ng ml21 and those of 2,6-TDA in the range 4.4–30 ng ml21 among the workers. In erythrocytes, the concentrations of 2,4-TDA were in the range 0.5–6.6 ng g21 and those of 2,6-TDA in the range 1.2–14 ng g21 among the workers. Significant linear relationships were seen between the mean concentrations, for all individual samples, in plasma and erythrocytes of 2,4-TDA (r = 0.97, n = 6) and 2,6-TDA (r = 0.97, n = 6) (Fig. 3). P-TDA declined slowly after work cessation. The half-lives in plasma (calculated as for UTDA) were in the range 6.5–12 d for 2,4-TDA (r = 0.76–0.95, n = 4) and 7–11 d for 2,6-TDA (r = 0.76–0.97, n = 4). E-TDA declined too little for the calculation of half-lives. When plotting the mean daily urinary concentrations adjusted for creatinine against P-TDA and E-TDA, relationships were observed. The relationships became more linear with the number of days after cessation of work (Fig. 4). Significant correlations were seen, with the exception of E-2,4-TDA on day 2. Significant linear plots, with the exception of E-2,6-TDA, were obtained on plotting the P-TDA and E-TDA for workers A, B, C, E and F against the TDI air levels measured on day 1 (worker D was only employed during the summer) (Fig. 5). No detectable P-TDA or E-TDA were found in the two volunteers, except for very low concentrations of 2,4-TDA ( < 0.33 ng ml21) in plasma from one of the volunteers.Among a group of 17 unexposed humans, the U-TDA and P-TDA were below the detection limit. Conditions for the Hydrolysis of Haemoglobin Volumes of 1 ml of the haemoglobin-containing supernatant (see Gel filtration of haemoglobin) were hydrolysed in 1.5 ml of 3 m H2SO4 at 100 °C for 4, 8, 16, 24 and 48 h. After hydrolysis the samples were subjected to the work-up procedure. No free 2,4- and 2,6-TDA were seen without hydrolysis.The release of 2,4- and 2,6-TDA increased with increasing hydrolysis time (Fig. 6). For the preparation of haemoglobin from the erythrocytes, the recoveries of 2,4- and 2,6-TDA were 77% and 91%, respectively. On analysing the remaining cell debris in the centrifuge tube no TDA was found. Gel Filtration of Proteins in erythrocytes TDA were only found in six of the 80 fractions (fractions 51–56) from the gel filtration separation and co-eluted com- Fig. 1 Concentrations of 2,4-TDA (2) and 2,6-TDA (5) in hydrolysed urine (mg per mmol of creatinine) from workers A–F.All urine produced during 2 work days and on the three days following work cessation (vacation), and on different occasions during the following 3–4 weeks, was collected. Time 0 = 6.00 am on the first day of the study. Analyst, January 1997, Vol. 122 53pletely with the haemoglobin (UV, 415 nm) peak. Fractions 51–56 contained 89% of the applied amounts of 2,4-TDA and 81% of 2,6-TDA (Fig. 7). In the GC–MS traces pure and well separated 2,4- and 2,6-TDA-PFPA peaks were seen. The 2,4-and 2,6-TDA-PFPA peaks in the chromatogram of fraction 54 were found at an unfiltered S/N of 175 and 61, respectively (rms). Only traces of TDA-PFPA were seen in fractions 1–50 and 57–80, due to impurities of TDA in the internal standards. These were also present in the chemical blanks. Discussion Isocyanates occur in many chemical and physical forms in workplace atmospheres. In the TDI foam industry, the monomers dominate in the gas phase.However, when particles are formed, many other compounds may be present in addition to monomers. These can be measured by methods which are based on the reaction of isocyanates with secondary amines containing a chromophore.14 During the thermal decomposition of PUR, in addition to isocyanates, aminoisocyanates and amines are also present. This means that isocyanate exposure measured by ‘conventional’ methods, underestimates the true exposure.It was therefore surprising, but encouraging, to find a relationship between the air data and the P-TDA and E-TDA levels among Fig. 2 Concentrations of 2,4-TDA (2) and 2,6-TDA (5) in hydrolysed urine (mg per mmol of creatinine) from two volunteers. The volunteers were present in the factory from 7 am until 4 pm on day 1. Time 0 = 6.00 am the first day of the study. Fig. 4 Concentrations of 2,4-TDA (5/-) and 2,6-TDA (2/8) in hydrolysed plasma (ng ml21) and erythrocytes (ng g21) and mean urinary concentration (mg per mmol of creatinine) during 1 d of exposure (day 2) and the three following exposure-free days (days 3–5) among six workers (A–F).Day 2: r P- 2,4-TDA = 0.84, r E-2,4-TDA = 0.81, r P-2,6-TDA = 0.86, r E-2,6-TDA = 0.83; Day 3: r P-2,4-TDA = 0.91, r E-2,4-TDA = 0.91, r P-2,6-TDA = 0.94, r E-2,6-TDA = 0.89; Day 4: r P-2,4-TDA = 0.92, r E-2,4-TDA = 0.98, r P-2,6-TDA = 0.97, r E-2,6-TDA = 0.98; Day 5: r P-2,4-TDA = 0.98, r E- 2,4-TDA = 0.97, r P-2,6-TDA = 0.99, r E-2,6-TDA = 0.97.Continuous line = 2,4-TDA; broken line = 2,6-TDA. Fig. 3 Mean concentrations, for all individual samples, of 2,4-TDA (5) and 2,6-TDA (-) in hydrolysed plasma (ng ml21) and erythrocytes (ng g21) among six workers (A–F) during 5 d. Continuous line = 2,4-TDA, r = 0.97; broken line = 2,6-TDA, r = 0.97. 54 Analyst, January 1997, Vol. 122the workers. However, the relationship was different to that for data from a flexible foam factory,11 where about the same levels in plasma were found but three times higher TDI air levels.This can be explained by the insufficiency and the limitations of the air methods used to measure more complex isocyanates. Data from conventional air methods may therefore be used as a relative index of exposure in the absence of methods that take into account all other isocyanates that may influence the industrial hygiene. Further studies of these aspects are in progress.The urine data clearly demonstrate fast elimination phases and one slower phase. It can be assumed that the fast phases reflect the more recent exposure and the slow phase reflects urinary elimination of degradation products of modified proteins, and hence the history of exposure. The urinary elimination half-lives for the slow phase were about the same as the half-life of albumin and the mean half-lives in plasma among TDI foam workers.11 The significant linear relationships between the U-TDA and E-TDA and P-TDA that improved with time after work cessation may also add to the explanation of the presence of modified erythrocytes and albumin metabolites and breakdown products excreted in urine.These products seem to dominate after a few days of work cessation. Worker D, a worker not chronically exposed, had shorter urinary elimination half-lives compared with the chronically exposed workers. The volunteers in this study had about the same urinary halflives as the volunteers in the volunteer study.12 The volunteer study may reflect an interesting difference in uptake, metabolism and excretion between chronically exposed workers and volunteers and the difference in exposure situations.In the workplace studies much more complex isocyanates and different chemical and physical forms are present in the air. Biomarkers of both 2,4- and 2,6-TDA were found in plasma and erythrocytes from all workers. The half-lives in plasma, in Fig. 5 Concentrations of 2,4-TDA (5) and 2,6-TDA (-) among workers A, B, C, E and F and in worker D (2 = 2,4-TDA) and (8 = 2,6-TDA) in: (a), hydrolysed plasma (ng ml21); and (b), hydrolysed erythrocytes (ng g21), plotted against the mean individual air concentrations of 2,4- and 2,6-TDI (mg m23) for the first work day of the study. For the calculation of the linear curves, data from worker D were excluded as he was a seasonal worker and not chronically exposed (r P-2,4-TDA = 0.98, r P- 2,6-TDA = 0.97, r E-2,4-TDA = 0.92, r E-2,6-TDA = 0.85).Fig. 7 Amounts of 2,4-TDA (5) and 2,6-TDA (~) (ng) in the fractions eluted from a gel filtration separation of lysed erythrocytes (cell debris separated) from worker A. Fractions 51–56 contained 89% of the applied amount of 2,4-TDA and 81% of 2,6-TDA and co-eluted with haemoglobin (UV 415 nm, broken line, 100% was saturated detection). Selected ion monitoring chromatogram of fraction 43 represents a fraction not containing TDA, and that of fraction 54 one with ‘TDI’-modified compounds.(m/z = 394.2 is the M 2 20 ions of TDA-PFPA and m/z = 397.2 is the M 2 20 ions of TDDA-PFPA). Fig. 6 Concentrations of 2,4-TDA (5) and 2,6-TDA (-) in lysed erythrocytes (cell debris separated) after hydrolysis for 0, 4, 8, 16, 24 and 48 h at 100 °C. Analyst, January 1997, Vol. 122 55this study, were about 50% shorter than those in the flexible foam worker study11 but about the same as those in the volunteer study.13 The difference may be explained by the presence of a faster phase, but this could not be observed in the study of flexible foam workers as blood samplings were performed at too long intervals. The E-TDA levels were virtually stable during the study and therefore no half-lives could be calculated.They can therefore be assumed to be longer than for plasma. A significant linear relationship between PTDA and E-TDA was observed. No TDA was found in the centrifuged and lysed erythrocyte solution on performing the work-up procedure without hydrolysis.This indicates covalently TDI-modified proteins. The gel filtration demonstrated that the modified protein in fact was haemoglobin. The hydrolysis pattern of haemoglobin was found to be almost the same as for plasma among TDI-exposed workers.7 This is an indication that the nature of the chemical bondings is the same and that the same hydrolysis conditions can be used. There are many aspects of the uptake, metabolism and excretion of isocyanates that are not yet fully understood. However, there are many reasons to believe that modified proteins and peptides are involved in isocyanate-associated disease. It is therefore interesting to know that intracellular in addition to intercellular proteins are modified with TDI among workers. The mechanism by which isocyanates are able to pass cell membranes is not known and further studies on this aspect are in progress. The present study has shown that the history of exposure can be determined by biomarkers in plasma and erythrocytes. Recent exposure does not greatly affect the P-TDA and E-TDA levels. Biomarkers in urine can be used for the same purpose if sampling is performed after the fast elimination phases. This work was supported by the Swedish Work Environment Fund. We thank M. Adamsson, M. Spanne and T. Russin for skilful technical assistance and Professor A. Grubb and Associate Professor J.-O. Jeppson for valuable discussions. References 1 Banks, D. E., Butcher, B. T., and Salviaggio, J. E., Ann. Allergy, 1986, 57, 389. 2 Baur, X., Marek, W., Ammon, J., Czuppon, A. B., Marczynski, B., Raulf-Heimsoth, M., Roemmelt, H., and Fruhman, G., Int. Arch. Occup. Environ. Health, 1994, 7, 310. 3 Vandenplas, O., Malo, J. L., Saetta, M., Mapp, C. E., and Fabbri, L. M., Br. J. Ind. Med., 1993, 50, 213. 4 Tinnerberg, H., Spanne, M., Dalene, M., and Skarping, G., Analyst, 1996, 121, 1101. 5 Skarping, G., Dalene, M., and Lind, P., J. Chromatogr., 1994, 663, 199. 6 Maitre, A., Berode, M., Perdrix, A., Romazini, S., and Savolainen, H., Int. Arch. Occup. Environ. Health, 1993, 65, 97. 7 Lind, P., Skarping, G., and Dalene, M., Anal. Chim. Acta, in the press. 8 Lind, P., Dalene, M., Lindstr�om, V., Grubb, A., and Skarping, G., Analyst, 1997, 122, in the press. 9 Sch�utze, D., Sepai, O., Lewalter, J., Miksche, L., Henschler, D., and Sabbioni, G., Carcinogenesis, 1995, 16, 573. 10 Persson, P., Dalene, M., Skarping, G., Adamsson, M., and Hagmar, L., Br. J. Ind. Med., 1993, 50, 1111. 11 Lind, P., Dalene, M., Skarping, G., and Hagmar, L., Occup. Environ. Med., 1996, 53, 94. 12 Skarping, G., Brorson, T., and Sang�o, C., Int. Arch. Occup. Environ. Health, 1991, 63, 83. 13 Brorson, T., Skarping, G., and Sang�o, C., Int. Arch. Occup. Environ. Health, 1991, 63, 253. 14 Tinnerberg, H., Dalene, M., and Skarping, G., Am. Ind. Hyg. Assoc. J., in the press. Paper 6/06148F Received September 6, 1996 Accepted November 6, 1996 56 Analyst, January 1997,