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ELSEVIER

J O U R NA L O IA NA L Y T l C A L a m l

Journal of Analytical and Applied Pyrolysis3 2 (1995) 127-136

A P P L I E D P Y RO L Y S I S

Determ ination of sulfur in biologically importantsubstances by pyrolysis-gas chromatographySeok Yu n Choi a, Ma n Goo Kim b, Hidenari Inoue .*

. Depurtm ent of Applied Chemistry, Keio Univ ersirJ. 3-14-1 ifi~mh i, Kohoku-k u. Yokohm m 223. Jupmrh Depcrr~ment of Environmenral Science, Kangweon Nat ional Unicersiry. 192-l. Hw jcr 2-Dong.

Chun chen Cit >, 200- 701. KorecrReceived I June 1994: accepted I5 August I994

AbstractAn analytical method for the determination of sulfur in biologically important substances

has been developed using pyrolysis-gas chromatograph y (Py-GC ) equipped w ith a flamephotometric 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 ofthe sulfur compounds contained in protein. The protein sample was pyrolyzed instanta-neously at high temperature (590C ) in a stream of nitrogen gas. The Py-GC methoddeveloped in the present study does not require any pretreatment and the analysis time fordetermining sulfur in biological samples is as quick as 30 min. The sulfur detection limit ofthis method was 40 ng for methionine and 60 ng for cystine and cysteine. The coefficient ofvariation was 5.7, 1 I and 1 4% for methionine, cystine and cysteine, respectively. Thedetermination of sulfur in biologically important substances such as huma n hair, dog hair.silk, etc. was carried out by the proposed PyyG C method. The analytical results revealedthat sulfur is 3.3-4.3 wt.% in huma n hair and 2.4-3.5 wt.% in dog hair.Kqw ords: Biological substances; C urie-point pyrolysis; Flame photometric detector: Gaschromatograph y; Hum an hair; Pyrolysis; Sulfur

* Corresponding author.

0165-2370/95 /$09.50 $2 1995 - Elsevier Science B.V. All rights reservedSSIII 0165-2370(94)00833-7

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1. IntroductionProtein substan ces are biologically important comp ounds in the natural world .

There have been various analytical meth ods for the determination of proteinsubstances, e.g. UV absorption spectrophotometry, fluorometry and ion chro-matography. How ever, these methods are tedious and time-consuming in thepretreatme nt of samp les. The developm ent of an accurate and conventional analyt-ical meth od is required for determining a small amount of protein substances withina short time. Gas chromatography was widely used to determine amino acids inprotein [ 11, althoug h it requires prederivatization of the amino acids. Recently thedetermination of amino acids has been advanced by improving the analyticalmethod and the analytical equipment. Lyle and Tehrani [2] reported a GC methodfor the determination of amino acids. Fujimaki et al. [3] and Ohsaw a et al. [4] haveintensively investigated a Py-GC meth od for the determination of sulfur-containingamino acids. In previous papers, Kim et al. [5,6] have applied the Py-CC methodto the determination of sulfur comp onents in air. In the present study we haveattempted to develop an accurate and conventional method for the determinationof sulfur in biologically important substances. The prop osed analytical meth od doe snot need any pretreatme nt of biological samp les because it is based on pyrolysis-gas chromatography equipped with a Curie point pyrolyzer and a sulfur-selectiveflame photometric detector.

2. Experimental

2.1. MaterialsAll chemicals were of reagent grade and used without further purification.

t_-Cystine and t_-methionine were purchased from Takara Kosan Co. and L-cysteinewas obtained from Junsei Chemical Co. The biologically important substancesinvestigated were human hair, dog hair, sheep hair, human nail and silk. Humanhair was taken from men in their 20 s and 30s and a nail sample was collected froma man in his 30s. Dog hair was taken from a Border collie, and 100% ) sheep hairand silk were obtained from Daiya Firm.

2.2. Pyrolysis -gas chromatog raphic conditionsA Curie-point pyrolyzer (Japan A nalytical Industry, JHP -2) was attache d directly

to a gas chromatograph (Shimadzu, GC-6AM ) equipped with a flame photometricdetector (FPD). The analytical column was a 3 m x 3 mm i.d. glass column packedwith 25% fi$-oxydipropionitrile coated on 60/80 mesh Chromoso rb W. Pyrolysis wascarried out at 590C for 6 s and a column oven w as maintained at 60C. Nitrogencarrier gas obtained from Nippon Sanso Co. was of higher than 99.9999% purity. Theregistered peak areas were measured by an integrator (Hitachi, D-2500) and given

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in integrator counts. The identification of pyrolysa tes was based on a compa risonof their chromatographic retention times with those of reference compounds.

2.3. Analvtical procedu resA series of standard stock solutions of sulfur (200 pg cm-) were prepared for

the calibration of a pyrolysis-gas chrom atogra ph using sulfur-containing aminoacids, i.e. L-methionine, L-cystine and L-cysteine. A sample of t_-methionine(93.02 mg), L-cystine (75.07 m g) and L-cysteine (76.05 m g) was weighed and dil-uted to 100 cm3 in a volumetric flask with distilled water. In the case of L-cystine10% aqueous ammonia was used in place of distilled water because this compoundis insoluble in water . S tandard solutions of sulfur in the concentrationrange 20-200 pg cmm3 were prepared by diluting a standard stock solutionwith distilled w ater. A sample o f the standard solution containing L-methionine,r.-cystine or L-cysteine was transferred on to a sheet of pyrofoil with a microsyringe. The pyrofoil was carefully heated on a hot plate at 60-C for l-2 min.After the solvent w as vaporized, the sample concentrated on the pyrofoil waswrapped and introduced into the Py--GC. Calibration graphs were constructed byrepeating these procedures and plotting the logarithm of peak intensities on thechromatogram vs. the logarithm of the amount of sulfur introduced into thePy-GC.

5E+3 400 500 600 700Temperature (C)

80 0

Fig. I. Effect of pyrolysis temperature on the yield of sulfur generated from methionine (??. cystine(0) and cysteine ( El).

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3. Results and discussion3.1. Pyr o l ys i s t emp er a t u r e

The yield of pyrolysis pro ducts of biologically impo rtant subs tance s is stronglydependent on pyrolysis temperature and pyrolysis technique. A series of pyrofoilswhich can cover the temperature range from 445 to 740C and a high-frequencyinduction technique were used in the present study. Pyrolysis was performed onsulfur-containing amino a cids, i.e. methionine, cystine and cysteine. The dependenceof the quantity of sulfur compoun ds generated on the pyrolysis temperature wasexamined by means of the sulfur-containing amino a cids. The results obtained fromthe above experiments are shown in Fig. 1 , where the yield of dimethyl sulfide isplotted as a function of pyrolysis tempe rature. The total peak area of pyrolysisproducts was also calculated from every sulfur compound generated at a pyrolysis

C H d H

A

io ;oRetentionhe (min)

B

io ioRetention timemill)

Fig. 2 (A and B).

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S. Y. Choi et al. 1 J. Anal. Appl. Pyrol~si.~ 32 (1995) 127 136 131

Ib 2bRctentfon t ime (mtn)

Fig. 2. Pyrograms observed at 590C with FPD. A, methionine: B. cystine; and C. cysteine.

temperature. As a result, it was determined that all the pyrolysis experiments shouldbe carried out at a pyrolysis temperature of 590C.3.2. Py rolys i s products o f b io logica l sam ples

The specific sulfur compounds in the pyrolysis products generated at 590C wereseparated on a column and selectively detected by a flame photometric detector(FPD ). Typical p yrogram s of sulfur-containing amino acids are show n in Fig. 2.Most peaks on the chromatogram consist of sulfur compounds such as hydrogensulfide, methyl mercap tan, dimethyl sulfide and carbon disulfide, which are easilygenerated by the pyrolysis of sulfur-containing amino acid residues [4]. An impor-tant pyrolysis product of methionine was methyl mercaptan, and the others werehydrog en sulfide and dimethyl sulfide. The pyrolysis produ ct typical of cystine andcysteine was hydrogen disulfide, and the others were methyl mercaptan, carbondisulfide and ethanethiol. The peak profile of pyrolysis produ cts from cystine and

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

Calibration graphs for the present pyrolysis-gas chromatographic method wereconstructe d using the standard solution p repared with methionine, cystine andcysteine. Methyl mercaptan and hydrogen sulfide were common compounds pro-duced by pyrolysis of methionine, cystine o r cysteine. Theref ore, a specific com-

A

C H S H

; ;oRetention time (min)

F i g . 3 ( A a n d B ) .

; lbRetention ime min)

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C

; lb0-m R e t e n t i o n t i m e ( m i n )

; li l ; IORetention rime (min) R e t e n t i o n t i m e ( m i n )Fig. 3 . Pyrograms of biologically important substances observed at 590C with FPD. A. human hair: B,human nail; C. dog hair; D, silk; and E, wool.

pound other than methyl mercaptan and hydrogen sulfide was searched for in orderto determine the quantity of sulfur in biological samp les. As a result, d imethylsulfide was used for methionine, and carbon disulfide for cystine and cysteine.How ever, it was impossible to detect carbon disulfide using the small amount ofstandard solutions containing cystine or cysteine. There fore, hydrog en sulfide wasutilized to determine sulfur in cystine and cysteine. Calibration graph s wer eobtained by plotting log (respon se) vs. log (weigh t), because nonlinearity is inherentto the FPDs response. The detection limit was determined as the point w here thecalibration curve deviated from a straight line. The detection limit for sulfur was 30ng according to our previous work [6], but in the present study it is 40 ng formethionine and 60 ng for cystine and cysteine. The coefficients of variation in thepresent experimen t are listed in Table 1. The large coefficients of variation for

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Table 1Coefficients of variation (C.V.) for sulfur-containing amino acids

Run Peak area (u)

Methionine Cystine Cysteine(CHsSCH,) (H,S) (HzS)

1 6.10 4.45 4.672 5.10 5.23 5.233 5.85 4.98 3.824 6.15 4.07 4.145 5.53 4.03 4.12Average 5.15 4.55 4.4S.D. (u) 0.33 0.5 0.61C.V. (%I) 5.7 11 14

Table 2Determination of sulfur in biologically important substances

Sample Methionine (wt.%) Cystine and cysteine (wt.%) Total sulfur (wt.%)

Human hair 1.10-1.23 2.21-3.08 3.3-4.3Human nail 1.24- .32 2.6992.81 3.994.1Dog hair 0.8551.17 1.58-2.35 2.4-3.5Silk 0.42-0.44 0.68%0.79 l.lL1.2Wool 0.4990.66 0.7440.85 1.2-1.5

cystine a nd cysteine are probably due to the poor reproducibility of FPD and theerror due to transferring the standard solution using a micro syringe.3.4. Determinat ion o f su l fur in b io logica l sam ples

Pyrolysis-gas chromatography equipped with an FPD was applied to the deter-mination of sulfur in biological samples. A fter the biological samples were washe dwith acetone, they were cut into pieces suitable for pyrolysis, wrapped in pyrofoiland subjected to Py-GC analysis. Pyrolysis experiments were repeated three timeswith each biological sam ple. Most of the biologically important substances gavetheir characteristic pyrolysis pattern when they were pyrolyzed at 590C andseparated under the above chromatographic conditions. The results of thedeterminations are summ arized in Table 2. Hum an hair contains 3.3-4.3% ofsulfur, do g hair 2.4-3.5%, and human nail 3.9-4.1%. The quantity of sulfur inhum an hair was slightly lower than that reported by Seta et al. [7] and Sak ada [S].The difference in assay methods may cause this disagreement in the analyticalresults on sulfur in human hair.

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S. Y. Choi 61 ul. 1 . And. A@. Pyolysi.r 32 (1995) 117 136

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 huma n hairThe pyrolysis-gas chromatograms of human hair sampled from individuals were

measured by the proposed Py-GC method. The peak area of the pyrolysis productswas normalized to the peak area of one pyrolysis product in particular. In otherwords, the ratio of the peak area of one particular pyrolysis product to those of theothers w as calculated. This technique is very useful in distinguishing betweenhuman hair samples of individuals. The human hair samples taken from two adultswere subjected to Py-GC analysis. After washing the samples w ith acetone, asuitable amount of hair samples was cut, weighed and wrapped with pyrofoil andintroduced into the Py-GC. Measurements of pyrograms of each human hairsample were repeated several times. The ratio of the peak area of hydrogen sulfideto those o f the other pyrolysis products was calculated and is illustrated in Fig. 4.The human hair samples taken from different persons gave the same types ofpyrolysis products, but the quantity of each pyrolysis component was different fromperson to person. The peak area of hydrogen sulfide, methyl m ercaptan or dimethylsulfide was chosen as a basis for the calculation of the peak-area ratio. Asatisfactory result was obtained for hydrogen sulfide, while the standard deviationwas large for methyl mercaptan and dimethyl sulfide. The normalized peak area isalmost invariant for the same person, but individuals have their own pattern in theratio of the peak area of pyrolysis products. This pyrolysis chromatographic feature

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of hum an hair could be used to identify a particular person or to distinguishbetween different persons in biochemistry and forensic medicine.

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