Journal

ELSEVIER

of Analytical and Applied 32 (1995) 127-136

Pyrolysis

JOURNALOI ANALYTlCALaml APPLIED PYROLYSIS

Determination of sulfur in biologically important substances by pyrolysis-gas chromatography

Seok Yun Choi a, Man Goo Kim b, Hidenari Inoue .*
.Depurtment ofApplied Chemistry, Keio UniversirJ. 3-14-1 ifi~mhi, Kohoku-ku. Yokohmm 223. Jupmr h Depcrr~ment of Environmenral Science, Kangweon National Unicersiry. 192-l. Hwjcr 2-Dong. Chunchen Cit>, 200- 701. Korecr Received

I June 1994: accepted

I5 August

I994

Abstract An analytical method for the determination of sulfur in biologically important substances has been developed using pyrolysis-gas chromatography (Py-GC) equipped with a flame photometric detector (FPD). This method is based on the determination of dimethyl sulfide (CH,SCH,) and hydrogen sulfide (H,S), each of which is one of the pyrolysis products of the sulfur compounds contained in protein. The protein sample was pyrolyzed instantaneously at high temperature (590C) in a stream of nitrogen gas. The Py-GC method developed in the present study does not require any pretreatment and the analysis time for determining sulfur in biological samples is as quick as 30 min. The sulfur detection limit of this method was 40 ng for methionine and 60 ng for cystine and cysteine. The coefficient of variation was 5.7, 1 I and 14% for methionine, cystine and cysteine, respectively. The determination of sulfur in biologically important substances such as human hair, dog hair. silk, etc. was carried out by the proposed PyyGC method. The analytical results revealed that sulfur is 3.3-4.3 wt.% in human hair and 2.4-3.5 wt.% in dog hair.
Kqwords: Biological substances; Curie-point pyrolysis; chromatography; Human hair; Pyrolysis; Sulfur

Flame

photometric

detector:

Gas

* Corresponding

author.

0165-2370/95/$09.50 SSIII

$2 1995 - Elsevier

Science B.V. All rights

reserved

0165-2370(94)00833-7

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32 (1995) 127

I36

1. Introduction Protein substances are biologically important compounds in the natural world. There have been various analytical methods for the determination of protein substances, e.g. UV absorption spectrophotometry, fluorometry and ion chromatography. However, these methods are tedious and time-consuming in the pretreatment of samples. The development of an accurate and conventional analytical method is required for determining a small amount of protein substances within a short time. Gas chromatography was widely used to determine amino acids in protein [ 11, although it requires prederivatization of the amino acids. Recently the determination of amino acids has been advanced by improving the analytical method and the analytical equipment. Lyle and Tehrani [2] reported a GC method for the determination of amino acids. Fujimaki et al. [3] and Ohsawa et al. [4] have intensively investigated a Py-GC method for the determination of sulfur-containing amino acids. In previous papers, Kim et al. [5,6] have applied the Py-CC method to the determination of sulfur components in air. In the present study we have attempted to develop an accurate and conventional method for the determination of sulfur in biologically important substances. The proposed analytical method does not need any pretreatment of biological samples because it is based on pyrolysisgas chromatography equipped with a Curie point pyrolyzer and a sulfur-selective flame photometric detector.

2. Experimental 2.1. Materials All chemicals were of reagent grade and used without further purification. t_-Cystine and t_-methionine were purchased from Takara Kosan Co. and L-cysteine was obtained from Junsei Chemical Co. The biologically important substances investigated were human hair, dog hair, sheep hair, human nail and silk. Human hair was taken from men in their 20s and 30s and a nail sample was collected from a man in his 30s. Dog hair was taken from a Border collie, and 100%) sheep hair and silk were obtained from Daiya Firm. 2.2. Pyrolysis -gas chromatographic conditions

A Curie-point pyrolyzer (Japan Analytical Industry, JHP-2) was attached directly to a gas chromatograph (Shimadzu, GC-6AM) equipped with a flame photometric detector (FPD). The analytical column was a 3 m x 3 mm i.d. glass column packed with 25% fi$ -oxydipropionitrile coated on 60/80 mesh Chromosorb W. Pyrolysis was carried out at 590C for 6 s and a column oven was maintained at 60C. Nitrogen carrier gas obtained from Nippon Sanso Co. was of higher than 99.9999% purity. The registered peak areas were measured by an integrator (Hitachi, D-2500) and given

S. Y. Choi et al. 1 J. Anal.

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Pyo1y.vi.v 3-1 (1995)

1-37~~136

I 20

in integrator counts. The identification of pyrolysates was based of their chromatographic retention times with those of reference 2.3. Analvtical procedures

on a comparison compounds.

A series of standard stock solutions of sulfur (200 pg cm- were prepared for ) the calibration of a pyrolysis-gas chromatograph using sulfur-containing amino acids, i.e. L-methionine, L-cystine and L-cysteine. A sample of t_-methionine (93.02 mg), L-cystine (75.07 mg) and L-cysteine (76.05 mg) was weighed and diluted to 100 cm3 in a volumetric flask with distilled water. In the case of L-cystine 10% aqueous ammonia was used in place of distilled water because this compound is insoluble in water. Standard solutions of sulfur in the concentration range 20-200 pg cmm3 were prepared by diluting a standard stock solution with distilled water. A sample of the standard solution containing L-methionine, r.-cystine or L-cysteine was transferred on to a sheet of pyrofoil with a micro syringe. The pyrofoil was carefully heated on a hot plate at 60-C for l-2 min. After the solvent was vaporized, the sample concentrated on the pyrofoil was wrapped and introduced into the Py--GC. Calibration graphs were constructed by repeating these procedures and plotting the logarithm of peak intensities on the chromatogram vs. the logarithm of the amount of sulfur introduced into the Py-GC.

5E+3 400 500 600 700 800

Temperature
Fig. I. Effect of pyrolysis temperature on the yield of sulfur

(C)
generated from methionine

( ?? cystine ).

(0) and cysteine ( El).

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3. Results and discussion 3.1. Pyrolysis temperature

The yield of pyrolysis products of biologically important substances is strongly dependent on pyrolysis temperature and pyrolysis technique. A series of pyrofoils which can cover the temperature range from 445 to 740C and a high-frequency induction technique were used in the present study. Pyrolysis was performed on sulfur-containing amino acids, i.e. methionine, cystine and cysteine. The dependence of the quantity of sulfur compounds generated on the pyrolysis temperature was examined by means of the sulfur-containing amino acids. The results obtained from the above experiments are shown in Fig. 1, where the yield of dimethyl sulfide is plotted as a function of pyrolysis temperature. The total peak area of pyrolysis products was also calculated from every sulfur compound generated at a pyrolysis

CHdH

io
Retention the
(min)

;o
Fig. 2 (A and B).

io
Retention

io
time (mill)

S. Y. Choi et al. 1 J. Anal. Appl. Pyrol~si.~ 32 (1995) 127 136

131

Ib

2b Rctentfon time (mtn)

Fig. 2. Pyrograms

observed

at 590C with FPD.

A, methionine:

B. cystine;

and C. cysteine.

temperature. As a result, it was determined be carried out at a pyrolysis temperature

that all the pyrolysis of 590C.

experiments

should

3.2. Pyrolysis products of biological samples The specific sulfur compounds in the pyrolysis products generated at 590C were separated on a column and selectively detected by a flame photometric detector (FPD). Typical pyrograms of sulfur-containing amino acids are shown in Fig. 2. Most peaks on the chromatogram consist of sulfur compounds such as hydrogen sulfide, methyl mercaptan, dimethyl sulfide and carbon disulfide, which are easily generated by the pyrolysis of sulfur-containing amino acid residues [4]. An important pyrolysis product of methionine was methyl mercaptan, and the others were hydrogen sulfide and dimethyl sulfide. The pyrolysis product typical of cystine and cysteine was hydrogen disulfide, and the others were methyl mercaptan, carbon disulfide and ethanethiol. The peak profile of pyrolysis products from cystine and

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32 (1995) 127-136

cysteine were fundamentally the same, and the quantity of each pyrolysis product also was almost equal. The pyrogram observed upon pyrolysis of biologically important substances is reproduced in Fig. 3. Most peaks on the chromatogram are the same as those generated from the pyrolysis of sulfur-containing amino acid residues. From these results it was confirmed that the biologically important substances of interest include sulfur-containing amino acid residues. 3.3. Calibration graphs of Py-CC

Calibration graphs for the present pyrolysis-gas chromatographic method were constructed using the standard solution prepared with methionine, cystine and cysteine. Methyl mercaptan and hydrogen sulfide were common compounds produced by pyrolysis of methionine, cystine or cysteine. Therefore, a specific com-

CHSH

;o
Fig. 3 (A and B).

lb

Retention time (min)

Retention time(min)

S. Y. Choi et al. / J. Anal. Appl. Pyrolysis 32 (1995) 127- 136

133

;
0-m

lb

Retention time (min)

;
Retention

lil
rime (min) observed

IO
hair: B,

Retention time (min)

Fig. 3. Pyrograms of biologically important substances human nail; C. dog hair; D, silk; and E, wool.

at 590C with FPD. A. human

pound other than methyl mercaptan and hydrogen sulfide was searched for in order to determine the quantity of sulfur in biological samples. As a result, dimethyl sulfide was used for methionine, and carbon disulfide for cystine and cysteine. However, it was impossible to detect carbon disulfide using the small amount of standard solutions containing cystine or cysteine. Therefore, hydrogen sulfide was utilized to determine sulfur in cystine and cysteine. Calibration graphs were obtained by plotting log (response) vs. log (weight), because nonlinearity is inherent to the FPD response. The detection limit was determined s as the point where the calibration curve deviated from a straight line. The detection limit for sulfur was 30 ng according to our previous work [6], but in the present study it is 40 ng for methionine and 60 ng for cystine and cysteine. The coefficients of variation in the present experiment are listed in Table 1. The large coefficients of variation for

134 Table 1 Coefficients Run

S. Y. Choi et al. 1 J. Anal. Appl. Pyrolysis 32 (1995) 127

I36

of variation

(C.V.)

for sulfur-containing

amino

acids

Peak area (u) Methionine (CHsSCH,) Cystine (H,S) 4.45 5.23 4.98 4.07 4.03 4.55 0.5 11 Cysteine (HzS) 4.67 5.23 3.82 4.14 4.12 4.4 0.61 14

1 2 3 4 5 Average S.D. (u) C.V. (%I)

6.10 5.10 5.85 6.15 5.53 5.15 0.33 5.7

Table 2 Determination Sample Human hair Human nail Dog hair Silk Wool

of sulfur

in biologically (wt.%)

important Cystine

substances and cysteine (wt. %) Total 3.3-4.3 3.994.1 2.4-3.5 l.lL1.2 1.2-1.5 sulfur (wt.%)

Methionine 1.10-1.23 1.24-I .32 0.8551.17 0.42-0.44 0.4990.66

2.21-3.08 2.6992.81 1.58-2.35 0.68%0.79 0.7440.85

cystine and cysteine are probably due to the poor reproducibility of FPD and the error due to transferring the standard solution using a micro syringe. 3.4. Determination of sulfur in biological samples Pyrolysis-gas chromatography equipped with an FPD was applied to the determination of sulfur in biological samples. After the biological samples were washed with acetone, they were cut into pieces suitable for pyrolysis, wrapped in pyrofoil and subjected to Py-GC analysis. Pyrolysis experiments were repeated three times with each biological sample. Most of the biologically important substances gave their characteristic pyrolysis pattern when they were pyrolyzed at 590C and separated under the above chromatographic conditions. The results of the determinations are summarized in Table 2. Human hair contains 3.3-4.3% of sulfur, dog hair 2.4-3.5%, and human nail 3.9-4.1%. The quantity of sulfur in human hair was slightly lower than that reported by Seta et al. [7] and Sakada [S]. The difference in assay methods may cause this disagreement in the analytical results on sulfur in human hair.

S. Y. Choi 6 ul. 1

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32 (1995)

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Fig. 4. Comparison

of the peak-area

ratios obtained for human hair of men in their 20s (A) and 30s (B).

3.5. Characterization

of human hair

The pyrolysis-gas chromatograms of human hair sampled from individuals were measured by the proposed Py-GC method. The peak area of the pyrolysis products was normalized to the peak area of one pyrolysis product in particular. In other words, the ratio of the peak area of one particular pyrolysis product to those of the others was calculated. This technique is very useful in distinguishing between human hair samples of individuals. The human hair samples taken from two adults were subjected to Py-GC analysis. After washing the samples with acetone, a suitable amount of hair samples was cut, weighed and wrapped with pyrofoil and introduced into the Py-GC. Measurements of pyrograms of each human hair sample were repeated several times. The ratio of the peak area of hydrogen sulfide to those of the other pyrolysis products was calculated and is illustrated in Fig. 4. The human hair samples taken from different persons gave the same types of pyrolysis products, but the quantity of each pyrolysis component was different from person to person. The peak area of hydrogen sulfide, methyl mercaptan or dimethyl sulfide was chosen as a basis for the calculation of the peak-area ratio. A satisfactory result was obtained for hydrogen sulfide, while the standard deviation was large for methyl mercaptan and dimethyl sulfide. The normalized peak area is almost invariant for the same person, but individuals have their own pattern in the ratio of the peak area of pyrolysis products. This pyrolysis chromatographic feature

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of human hair could be used to identify a particular person or to distinguish between different persons in biochemistry and forensic medicine.

References
[I] [2] [3] [4] [5] [6] [7] [8] M.A. Posthumun and N.M.M. Nibbering, Org. Mass Spectrom., 12 (1977) 334. S.J. Lyle and MS. Tehrani, J. Chromatogr., 240 (1982) 209. M. Fujimaki, S. Kato and T. Kurata, Agric. Biol. Chem., 33 (1969) 1144. M. Ohsawa, H. Ohtani and S. Tsuge, Fresenius Z. Anal. Chem., 329 ( 1988) 781. M.G. Kim, K. Yagawa, H. Inoue and T. Shirai, Bunseki Kagaku, 38 (1988) 233. M.G. Kim, K. Yagawa, H. Inoue and T. Shirai, J. Anal. Appl. Pyrolysis, 20 (1991) 263 S. Seta, H. Sato and M. Yoshino, First Medico-Legal Sec., NIPS, 31 (1978) 32. S. Sakada, Medical J. Hiroshima Univ., 4 (1956) 1357.