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Metabolite Determination

Determination of a bound musk xylene metabolite in carp hemoglobin as a biomarker of exposure by gas chromatography mass spectrometry using selected ion monitoring

Published in J. Anal. Toxicol., 2004.
[note: minor content and formatting differences exist between this web version and the published version]

M. A. Mottaleb*, W. C. Brumley, S. M. Pyle and G. W. Sovocool

1U.S. Environmental Protection Agency, Environmental Sciences Division, P.O. Box 93478, Las Vegas, NV 89193-3478

*Corresponding author: Fax: (702) 798 2142, Email: mmottaleb@yahoo.com


Musk xylene (MX) is widely used as a fragrance ingredient in commercial toiletries. Identification and quantification of a bound 4-amino-MX (AMX) metabolite was carried out by gas chromatography-mass spectrometry (GC/MS), with selected ion monitoring (SIM). Detection of AMX occurred after the cysteine adducts in carp hemoglobin, derived from the nitroso metabolite, were released by alkaline hydrolysis. The released AMX metabolite was extracted into n-hexane. The extract was preconcentrated by evaporation, and analyzed by GC-SIM-MS. The concentration of AMX metabolite was found to range from 6.0 to 30.6 ng/g in the carp Hb, collected from the Las Vegas Wash and Lake Mead, Nevada areas. The presence of an AMX metabolite in the carp Hb was confirmed when similar mass spectral features and the same retention time of the AMX metabolite were obtained for both standard AMX and carp Hb extract solutions. In the non-hydrolyzed and reagent blank extracts, the AMX metabolite was not detected.


Musk xylene (MX) and other synthetic musks are used as synthetic fragrances in formulations of cleaners, soaps, detergents and body-care products. The commercial and domestic use and discharge of these compounds into municipal sewage systems has led to their ubiquitous occurrence in the aquatic environment and their presence at various concentrations in some organisms of certain aquatic ecosystems (1). Due to the low extent of biodegradation and high lipophilicity (2), MX and synthetic musks have high potential as environmental contaminants and are capable of being bioconcentrated in aquatic and terrestrial organisms (3). In particular, the occurrence of MX in an aquatic organism was first reported in 1981 (4). Since then, MX has been detected in most environmental compartments: in the North Sea, rivers and freshwater (5-7), domestic and industrial sewage sludge (8), sewage treatment effluent (9), Norwegian air samples (10), human adipose tissue and breast milk (2, 11), developing and adult rats (12), fish, mussels, and shrimp (1), and whole fish (13). Due to the ubiquitous distribution, and bioaccumulation potential of nitro musks in the environment, MX was banned in Japan in the 1980s. In Europe, MX and musk ketone are under discussion (5) because of their environmental and toxicological impact.

Figure 1 shows the metabolic path of MX conversion to AMX. MX and its metabolite have been identified and quantified in samples of domestic and industrial sewage waters (14). However, despite widespread use and occurrence of MX, only limited information about its toxicology is available. The acute toxicity of MX is low, although a non-dose-dependent increase in the incidence of liver tumors was reported in mice after long-term administration of MX in the diet (15). A half-life for elimination for MX of less than a few days is found in the rat; however, this contrasts with a slow elimination and half-life of about 80 days in humans (16). MX is known as a co-mutagenic substance for a great number of polycylic aromatic compounds and aromatic amines. MX was identified as an inducer of toxifying enzymes in rat liver, and it is a cytochrome P450 1A2 isoenzyme inducer (17).

Figure 1. Metabolic path of hemoglobin cysteine adduct formation with nitro musk. Biotransformation of musk xylene (A) to 4-amino-MX (B).

The measurement of various biomarkers is used to indicate exposure to a variety of xenobiotics. Biomarkers have been found in urine, saliva, and hair, as well as serum proteins. One of the most promising biomarkers of exposure to xenobiotics involving nitroaromatics is the blood protein hemoglobin. Nitroaromatics and their metabolites form adducts with hemoglobin (Hb). The binding of metabolites from nitroarenes to hemoglobin may be used as a marker of exposure, which has resulted from the passage of the xenobiotic through one or more barriers of the organism (e.g., skin, intestinal mucosa and lung mucosa). Hemoglobin-bound metabolites as biomarkers of exposure may be used to assess cumulative exposures over a longer time range (the life time of red blood cells) and thus may be better suited for risk assessment than quantitation of urinary metabolites (18,19). Nitroarenes are enzymatically reduced and their metabolites, nitrosoarenes, react with the sulfhydryl group of cysteine in hemoglobin to form an acid/base labile sulfinamide that hydrolyzes to aromatic amines in the presence of aqueous base. The metabolic path of MX and Hb cysteine adduct formation is shown in Figure 1. It is reported that the hemoglobin adduct of an aromatic amine is a good dosimeter for the target tissue dose of the ultimate carcinogenic metabolite of the amine (19). The biotransformation and toxicokinetics of MX metabolites in human hemoglobin have been reported (20,21). Although there are studies conducted for the detection of an AMX metabolite in different environmental compartments (1-21), to our knowledge no work has been performed on the determination of AMX metabolites from carp hemoglobin for the purpose of ecological assessment of MX exposure. This paper describes the determination of a bound AMX metabolite, formed by enzymatic reduction of MX, followed by adduct formation with the carp hemoglobin sample, collected from the Las Vegas Wash (LVW) and Lake Mead (LM), Nevada. The hemoglobin adducts are detected and quantified by gas chromatography mass spectrometry (GC-MS) with selected ion monitoring (SIM).

Materials and method

Reagents and chemicals

Sodium dodecyl sulfate (SDS), sodium hydroxide pellets, and n-hexane (HPLC grade) were obtained from Sigma-Aldrich, Fisher Scientific, and J. T. Baker, respectively. The internal standard (I.S.), Naphthalene-d8, was purchased from Absolute Standard Inc., CT. The standard solution of 4-amino-MX (AMX) metabolite was obtained gratis from Dr. L. I. Osemwengie, U.S. Environmental Protection Agency, Las Vegas, Nevada. Solutions with known amounts of metabolite and I.S. were used to prepare calibration curves to quantify the results. De-ionized water was used for all preparations where necessary.

Collection of carp blood samples

Live carp were obtained from downstream (about 100 m distance) of a publicly owned wash, called Las Vegas Wash, and from LM, Nevada. The domestic and sewage water treated by public sanitation treatment plants (STP) flowed down through a canal to LM. The distance between the LVW and LM sample collection points was about 10 miles. About 6.5 to 8.0 mL of fresh blood samples were collected in whole blood tubes containing heparin solution (Daigger and Company Inc., IL). Blood was obtained from the carp by two methods, (i) severing the caudal peduncle (SCP; used for smaller carp) and /or (ii) using cardiac puncture. After collection of blood, the tubes were shaken and placed on ice.

Isolation of hemoglobin

Fresh, heparinized blood samples were centrifuged at 3000 x g for 10 min at 4oC and the red blood cells (RBC) were separated from the plasma. The RBC were suspended/washed two times with equal volumes of freshly prepared 0.9% NaCl solution. The cells were lysed by addition of 2 volumes of distilled water, and the solutions were centrifuged again at 3000 x g for 10 min at 4oC. The cellular debris was discarded after centrifugation. The hemoglobin solutions were dialyzed for 72 h to remove small molecules and then placed in a freezer to freeze the hemoglobin solution. A freeze-drying procedure was employed to eliminate water from the frozen Hb by a Sentry Microprocessor Control, Freezemobile and Benchtop Freeze-dryer (The VirTis Company, Inc. NY). The dried Hb was then placed in a freezer for later analysis of the nitro musk metabolite.

Alkaline hydrolysis

To release the bound amino metabolite from the carp hemoglobin, alkaline hydrolysis was performed. The detailed descriptions of basic hydrolysis, extraction, and preconcentration procedures were described in our earlier work (22). Briefly, about 13 to 76 mg of dried hemoglobin were placed in cleaned and dried tubes, followed by 9 mL of 0.5% SDS solution and 1 mL of 10 N NaOH solution. The pH of the solution was determined to be 12. The mixture was then stirred for 1 h at room temperature and extracted 3 times with 10 mL of n-hexane. The tube was placed in a refrigerator for about 45 min to freeze the aqueous sample. A clear hexane layer was obtained as an extract on the top of the aqueous layer in the tube. The residual water from the extract was removed by passing the extract through a drying column containing granular anhydrous Na2SO4. The dried extract was then concentrated by evaporation under a stream of nitrogen. The I.S. was added, the solution sealed in GC-vials, and the sample analyzed by GC-SIM-MS. A schematic representation of the experimental procedure is shown in Figure 2.

Figure 2. Schematic representation of experimental procedure.

Non-hydrolyzed and reagent blank experiments

To investigate whether any unbound AMX metabolite was present in the carp Hb, a control experiment without basic hydrolysis was carried out. In this experiment, except for the NaOH, all chemicals and solvents were added to the Hb and the same extraction and proconcentration procedures were followed as described in the hydrolysis work. A laboratory or reagent blank control experiment was also performed by taking the same amounts of solvents, chemicals, and reagents used for the hydrolysis experiment, except the hemoglobin.

Gas chromatography and mass spectrometry

An Agilent Technologies HP 6890 series GC system equipped with a HP 5973 mass selective detector (MSD) connected to a Agilent 7683 auto sampler and Agilent 6890 GC were used. The helium carrier gas was passed through a DB-5 (J&W Scientific, Agilent Technologies, CA) capillary column (40 m long, 0.180 mm i.d., and 0.18 mm film thickness) at a constant flow rate of 0.5 mL/min (average linear velocity 22 cm/sec) using the pulsed splitless mode. The auto sampler injected a 2-mL volume of sample or standard solution into the GC with gradient oven temperature starting at 60oC for 1 min, 150oC at 10oC min-1, 250oC at 8oC min-1, and 300oC at 10oC min-1, and holding the final temperature for 6 min. The injector and transfer line temperatures were 250 and 280oC, respectively. The ion source temperature was 230oC and operated in the 70 eV electron ionization (EI) mode. By selecting base peak and confirming ions of the I.S. and the target compounds, the mass spectral acquisitions were performed with dwell times of 25 msec/ion using the GC/MSD Agilent ChemStation software, version B.02.05. In the case of the I.S (naphthalene-d8), the base peak ion m/z 136 was also the molecular ion, and for the target compound (AMX), the base peak ion was m/z 252 (M-15) with five confirming ions at m/z 268, 267 (molecular ion), 253, 218, and 215 selected for monitoring.

Calibration Curve

A regression analysis was carried out on the ratio of areas (analyte area divided by internal standard area) versus the ratio of AMX concentration to internal standard concentration resulting in a 6-point calibration curve. Unweighted regression was considered and resulted in an R2 of 0.998 for forcing the equation through zero signal at zero concentration and also with no forcing through zero signal. The calibration curve resulting from forcing the equation through the point 0,0 was employed to determine the AMX in hemoglobin samples because the concentration of AMX was expected to be near zero. The resulting equation of the line was used to calculate the concentrations of AMX in the samples and check standards run during the course of analysis. The concentration of AMX was calculable based on the known volume and concentration of internal standard added to the sample and the known mass of the sample being analyzed.

Quality Assurance/Quality Control (QA/QC)

Each group of samples to be analyzed was bracketed before and after by a representative standard/internal standard QC sample to establish adherence to the calibration curve equation and agreement with the retention time of the standard. Deviations from the calibration curve greater than 10% would cause rerunning of standards, construction of a new calibration curve, or replacement of the capillary GC injector as a result of poor peak shape (tailing), which affected quantitation. Retention time variations were generally less than 0.08 % and peak widths at half-height were about 3 sec.

A laboratory reagent blank was analyzed using the same procedure as was used for the hemoglobin samples, except no hemoglobin was extracted. No AMX was detected as a background contaminant. Standards run on the day of analysis were followed by a solvent blank run, followed by the extract. No carryover of AMX was observed.

Confirmation of identity was based on the presence of six ions whose relative abundances agreed to within 20 % of the relative abundances of the standard, and whose retention time was within 2 sec of that of the standard.

Results and discussion

The alkaline hydrolysis, non-hydrolyzed, and reagent blank experiments were performed to obtain and support the identification and quantification of the AMX metabolite as truly coming from the carp hemoglobin. In the alkaline hydrolysis, the MX bound to carp hemoglobin was released as the AMX metabolite that was extracted into n-hexane. It was considered that the unbound AMX metabolite might also be present in the carp hemoglobin. To investigate the possible presence of unbound AMX metabolite, the non-hydrolyzed and reagent blank experiments were also performed. The preconcentrated extract obtained from the non-hydrolyzed and reagent blank experiments were spiked with a known concentration of I.S. solution. The AMX metabolite standard solution (50 pg/mL) containing 200 pg/mL I.S. was prepared. The standard, solvent blank, non-hydrolyzed Hb extract, solvent blank, and reagent blank extract were injected into the GC-MS in this order. A 2-mL volume of each solution was injected into the GC by the autosampler. In the case of the standard solution, it was observed that the I.S. and AMX metabolite eluted from the GC capillary column at 11.19 and 24.43 min, respectively, and the appropriate base peak and confirming ions, in the correct relative intensity ratios, were also observed at these times. The mass chromatograms and the spectra of the standard AMX metabolite will be shown in the detection of a bound AMX metabolite in the carp hemoglobin section below. When non-hydrolyzed and reagent blank extracts were considered, the GC-MS chromatograms did not show any peak for AMX at 24.43 min and no mass signals of the ions for AMX metabolite were observed. This indicated that no unbound AMX metabolite was present in the carp hemoglobin investigated in this study. This further supports the inference that AMX results from liberation by hydrolysis from the Hb-bound metabolite (21).

Detection of a bound AMX metabolite in the carp hemoglobin

To determine the AMX metabolite in the carp hemoglobin, the preconcentrated extracts obtained from the hydrolyzed experiments were spiked with a known concentration of I.S. solution. A standard solution (50 pg/mL) of AMX metabolite containing 200 pg/mL I.S. was also prepared. A 2-mL volume each of standard, solvent blank, and hydrolyzed Hb extract was injected in this order into the GC-MS by the autosampler. The same base peak and confirming ions were monitored for identification of the I.S. and AMX metabolite as were monitored for the non-hydrolyzed extract. The AMX metabolite was found in the hydrolyzed extract solution. Figure 3 shows an overlay of mass chromatograms of selected m/z 267, 252, and 218 ions obtained from (A) standard solution of AMX metabolite and for (B) hydrolyzed carp Hb extract for detection of AMX metabolite. These show a similarity within 20% of the selected ion relative abundances for the AMX metabolite eluted from the capillary column at a retention time of 24.43 min.

Figure 3. Overlay of GC-SIM-MS chromatograms for (A) standard solution (50 pg/mL) of AMX containing 200 pg/mL I.S (naphthalene-d8) and (B) sample extract solution (extract of 76.1 mg Hb collected from the LVW carp) containing 400 pg/mL I.S. Injection volume 2 mL, capillary column DB-5 (40 m long, 0.180 mm i.d., and 0.18 mm film thickness). GC-MS operation conditions are given in the experimental section.

By selecting the peaks in the selected ion monitoring chromatograms of the standard and sample extract solutions and then subtracting the background level by ChemStation software, the mass spectra were produced. Figure 4 shows the mass spectra for (A) standard AMX solution and (B) for sample extract solution. The mass spectra are only shown between the ranges of m/z 210 and 270 ions because the base peak and confirming ions for the identification of the AMX metabolite were selected in this range. In general, the mass spectral features compared very well to each other and indicate the presence of bound AMX metabolite in the carp hemoglobin. A small difference of relative abundances could be observed in some confirming ions relative to the standard. This could be a matrix effect on the ion levels detected.

Figure 4. GC-MS derived SIM mass spectra for (A) standard AMX metabolite, taken from peak at 24.43 min from the Figure 3 (A), and (B) AMX metabolite detected in carp Hb sample solution, taken from peak at 24.43 min from the Figure 3 (B). Conditions are same as in Figure 3.

Quantification of the AMX metabolite in the carp hemoglobin samples

The AMX metabolite previously bound to the carp hemoglobin cysteines was detected. A GC-MS response calibration curve for AMX quantification in the carp hemoglobin was prepared by injecting a series of standard solutions (1.0, 10.0, 25.0, 50.0, 100.0 and 200 pg/mL) of AMX containing I.S. (200 pg/mL) into the GC-MS instrument. The linear calibration curve fitted the experimental data with an R2 of 0.998. Using the ChemStation software, the concentration of the AMX metabolite liberated from the carp hemoglobin was calculated. Table 1 summarizes the concentrations of AMX metabolite that were obtained from the alkaline hydrolysis, non-hydrolyzed, and reagent blank experiments. Due to the limited amount of the carp hemoglobin samples, only a few hydrolyzed extractions were performed. The extraction performed with the hemoglobin (13.3 to 76.1 mg) gave concentration levels of AMX metabolite ranging from 6.0 to 30.6 ng/g. In the case of LVW samples, the 19.8 or 13.3 mg Hb samples that released the AMX metabolite by hydrolyzed extraction, were about 4 or 5 times lower than the amount obtained from the 76.1 mg Hb sample. This may be explained by the fact that the smaller sample weights of 13.3 or 19.8 mg, taken for hydrolyzed extraction, liberated small amounts of AMX metabolite that were near the GC-MS detection level. Another possibility is sample inhomogeneity because the carp hemoglobin was extracted from about 6.5 to 8 mL of carp blood and after freeze-drying, the dried hemoglobin samples were not homogenized.


The binding of AMX metabolite and cysteine in Hb has been detected in carp by GC-SIM-MS. The reduction of a nitro group in MX yielded an intermediate capable of forming an adduct of Hb that could yield an amine that would be suitable as a biochemical endpoint, useful for monitoring and assessment of MX hazards exposure. The concentration of bound AMX metabolite was found in the carp Hb in the range of 6.0 to 30.6 ng/g. The use of hemoglobin adducts as biomarkers for nitro musk exposure in fish populations appears to be worthy of further study.


This work was performed while the author (MAM) held a National Research Council Research Associateship Award at the National Exposure Research Laboratory, U.S. Environmental Protection Agency (EPA), Las Vegas, Nevada. MAM would like to thank the Department of Chemistry, University of Rajshahi, Bangladesh for granting study leave to perform the research in the EPA laboratory.

Notice: The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded and performed the research described. This manuscript has been subjected to the EPA's peer and administrative review and has been approved for publication. Mention of trade names or commercial products in the manuscript does not constitute endorsement or recommendation by EPA for use.


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Table 1. Concentration of AMX metabolite in the hemoglobin obtained from the Las Vegas Wash and Lake Mead carp blood samples.

Live carp samples collection points Experiments performed for extraction of AMX Amount of Hemoglobin extracted (mg) Quantified ions Concentration of AMX metabolite (ng/g)
Las Vegas Wash (LVW) Hydrolyzed 76.1 252 30.6
LVW Hydrolyzed 19.8 252 8.2
LVW Hydrolyzed 13.3 252 6.0
LVW Non-hydrolyzed 43.6 252 ND
Lake Mead (LM) Hydrolyzed 30.1 252 14.8
LM Hydrolyzed 30.6 252 16.2
LM Non-hydrolyzed 32.5 252 ND
*Reagent or laboratory blank - - 252 ND

Reagent blank experiment was performed by taking the same amount of reagents used for the hydrolysis experiment, except hemoglobin. ND represents not detected.

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