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Forensic Science International 157 (2006) 134–143 www.elsevier.com/locate/forsciint
Identification of inorganic anions by gas chromatography/mass spectrometry Masataka Sakayanagi a,*, Yaeko Yamada b, Chikako Sakabe b, Kunio Watanabe a, Yoshihiro Harigaya b a
Scientific Criminal Investigation Laboratory, Kanagawa Prefectural Police Headquarters, 155-1 Yamashita-Cho, Naka-Ku, Yokohama 231-0023, Japan b School of Pharmaceutical Science, Kitasato University, 5-9-1 Shirokane Minato-ku, Tokyo 108-8641, Japan Received 25 February 2005; accepted 8 April 2005 Available online 10 May 2005
Abstract Inorganic anions were identified by using gas chromatography/mass spectrometry (GC/MS). Derivatization of the anions was achieved with pentafluorobenzyl p-toluenesulphonate (PFB-Tos) as the reaction reagent and a crown ether as a phase transfer catalyst. When PFB-Br was used as the reaction regent, the retention time of it was close to those of the derivatized inorganic anions and interfered with the analysis. In contrast, the retention time of PFB-Tos differed greatly from the PFB derivatives of the inorganic anions and the compounds of interest could be detected without interference. Although the PFB derivatives of SO4, S2O3, CO3, ClO4, and ClO3 could not be detected, the derivatives of F, Cl, Br, I, CN, OCN, SCN, N3, NO3, and NO2 were detected using PFB-Tos as the derivatizing reagent. The inorganic anions were detectable within 30 ng approximately, which is of sufficient sensitivity for use in forensic chemistry. Accurate mass number was measured for each PFB derivative by high-resolution mass spectrometry (HRMS) within a measurement error of 2 millimass units (mmu), which allowed determination of the compositional formula from the mass number. In addition, actual analysis was performed successively by our method using trial samples of matrix. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic science; Inorganic anion; Pentafluorobenzyl derivatives; Crown ether; Gas chromatography; Mass spectrometry
1. Introduction The analysis of inorganic anions in forensic chemistry is important for the analysis of powder residue from explosives and contamination of poisons. Generally, analysis of inorganic anions is accomplished by ion chromatography [1,2]. Recently, capillary electrophoresis methods [2–8] have been developed.
* Corresponding author. Tel.: +81 45 662 0395; fax: +81 45 662 0395. E-mail address: [email protected] (M. Sakayanagi).
Ion chromatography and capillary electrophoresis identify inorganic anions by retention time or migration time. However, a sample contaminated with matrix is often difficult to analyze. Gas chromatography/mass spectrometry (GC/MS) identifies compounds by chromatography retention time and mass spectrum, making the method a highly accurate procedure and an essential method in forensic chemistry. Inorganic anions are not volatile, so analysis by gas chromatography directly is difficult. Therefore, GC methods for inorganic ions require conversion to a volatile derivative such as a methyl [9], ethyl [10], butyl [11], or pentafluorobenzyl (PFB) derivative [12–16]. Among them, PFB derivatives have been used for supersensitive ECD
0379-0738/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2005.04.003
M. Sakayanagi et al. / Forensic Science International 157 (2006) 134–143
(electron capture detector) in GC [12] and GC/MS [13–16]. The conventional synthesis of PFB derivatives of inorganic anions uses a quaternary ammonium salt as a phase transfer catalyst, with the reaction conducted between an organic (salt of anion) and an aqueous (PFB-Br and PFB derivative of anion) phase. Funazo et al. [12] reported that bromide, iodide, cyanide, thiocyanate, nitrite, nitrate, and sulfide could be converted to PFB derivatives using pentafluorobenzyl p-toluenesulphonate (PFB-Tos) as the derivatizing reagent and tetra-n-amylammonium chloride as a phase transfer catalyst. The PFB derivatives were analyzed by GC with a flame ionization detector. But this method was not ideal for the identification of unknown ions, because GC discriminated among unknown agents only by retention time. Kage et al. reported the analysis of PFB derivatives of inorganic anions by GC/MS. Cyanide [13], thiocyanate [13], nitrite [14], nitrate [14], and azide [15] were converted to PFB derivatives using pentafluorobenzyl bromide (PFB-Br) and tetra-decyldimethylbenzylammonium chloride (TDMBA) as a phase transfer catalyst. In addition, Tsuge et al. [16] reported that cyanide, thiocyanate, nitrite, nitrate, azide, and sulfide could be converted to PFB derivatives using PFB-Br as the derivatizing reagent and TDMBA or polymer-bound tributylmethylphoshonium chloride as a phase transfer catalyst, and then identified by GC/MS. However, when the quaternary ammonium salt was used as the phase transfer catalyst as shown in the case of Kage et al. and Tsuge et al., the anion of the salt (e.g., chloride or bromide ion) was present in the reaction mixture. Therefore, the detection of chloride or bromide was difficult. This study was designed to develop a method for the discrimination of inorganic anions under a single condition. Crown ethers were used as a phase transfer catalyst because they do not contain inorganic anions. The inorganic anions were derivatized between a solid (salt of anion) and liquid (PFB-Tos and PFB derivative of anion) phase by PFB-Tos, using a crown-ether as the phase transfer catalyst. This resulted in a more convenient reaction process than the previously reported processes. PFB derivatives were detected by GC/MS, and detection limits were determined. Furthermore, the application of our method to trial samples of matrix was actually performed by using beverages which contained azide and cyanide; because, sodium
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azide and potassium cyanide become a problem in matters relating to poisoning in Japan. For low-resolution mass spectrometry (LRMS), the same mass number can indicate different compositions. Because LRMS determines integral mass; it cannot unambiguously identify a compound. Then, accurate mass measurement by high-resolution mass spectrometry (HRMS) becomes important. Accurate mass measurement of PFB derivatives of inorganic anions to identify compositional formula using highresolution GC/MS has not yet been reported. Therefore, we determined the compositional formula of PFB derivatives of inorganic anions by measurement of the mass number.
2. Experimental 2.1. Materials 2.1.1. Inorganic anions Inorganic anions were purchased from Wako Pure Chemical Industries Co. Ltd. (Osaka, Japan) and Kanto Chemistry Co. Ltd. (Tokyo, Japan). The following reagents were used: potassium fluoride (KF), sodium chloride (NaCl), sodium bromide (NaBr), sodium iodide (NaI), potassium cyanide (KCN), potassium cyanate (KOCN), potassium thiocyanate (KSCN), sodium azide (NaN3), potassium nitrate (KNO3), sodium nitrite (NaNO2), potassium perchlorate (KClO4), sodium chlorate (NaClO3), sodium sulfate (Na2SO4), sodium thiosulfate (Na2S2O3), and sodium carbonate (Na2CO3). 2.1.2. Derivatizing reagent Pentafluorobenzyl p-toluene sulfonate (PFB-Tos, TCI; Tokyo, Japan) was used for the derivatizing regent. PFB-Tos (1.761 g) was dissolved in 100 ml of dichloromethane to give a concentration of 50 mM. 2.1.3. Phase transfer catalysts The 18-crown-6-ether (18-crown-6, Wako Pure Chemical Industries Co. Ltd., Osaka, Japan) and 15-crown-5-ether (15-crown-5, Aldrich; St. Louis, MO, USA) were used as phase transfer catalysts. 18-crown-6 (264.3 mg) and 15crown-5 (220.2 mg) were dissolved in 100 ml of dichloromethane to give a concentration of 10 mM.
Fig. 1. Formula for the PFB derivatives.
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The salts of inorganic ions are usually insoluble in organic solvents. However, cations such as sodium and potassium become trapped in the cavity of some crown ethers that act as catalysts. A segregated anion with high reactivity is formed
with the crown ether and becomes soluble in organic solvents. 15-crown-5 chelates sodium ions, while 18-crown-6 chelates potassium and ammonium ions. The other regents used were of analytical grade.
Fig. 2. Mass chromatograms of pentafluorobenzyl inorganic anion derivatives.
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2.2. General procedure for PFB derivatization A 0.01 mmol inorganic anion, 0.2 ml volume of 50 mM PFB-Tos solution, 0.1 ml of 10 mM crown ether solution, and 0.9 ml of dichloromethane were put into a 5 ml volume of glass vial for derivatization. After the mixture was stirred at room temperature for 90 min, it was diluted with dichloromethane, and injected into the GC/MS. Fig. 1 outlines the PFB derivatization of inorganic anions. 2.3. GC/MS conditions GC/MS analysis was performed with a Hewlett-Packard HP-5890II GC instrument (Palo Alto, CA, USA) equipped
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with a JEOL JMS-AX505HA double-focusing mass spectrometer (Tokyo, Japan). The GC was equipped with an HP-1MS-fused silica capillary column (30 m 0.25 mm inside diameter, 0.25 mm film thickness). Temperature was programmed from 45 8C (1.5 min hold) to 280 8C at 10 8C/ min. Helium was used as the carrier gas. The injection port was operated in splitless mode and valve off time was set at 1 min. GC injection port and transfer line temperatures were 250 and 300 8C, respectively. GC injection port pressure was 11 psi. Ionization energy of positive-ion electron impact ionization (EI) was 70 eV. Ionization energy and the reagent gas of the negative-ion chemical ionization (CI) conditions were 200 eVand isobutene, respectively. The ionizing current was measured at 300 mA. HRMS was
Fig. 3. Mass spectra of pentafluorobenzyl inorganic anion derivatives. (A) Positive EI mass spectrum of PFB-F, (B) positive EI mass spectrum of PFB-Cl, (C) positive EI mass spectrum of PFB-Br, (D) positive EI mass spectrum of PFB-I, (E) positive EI mass spectrum of PFB-CN, (F) positive EI mass spectrum of PFB-OCN.
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conducted by the electric field scanning method, and LRMS was conducted by the magnetic field scanning method. HRMS experiments were performed at a resolution of 3000 (10% valley definition), and LRMS experiments were performed at a resolution of 500 (10% valley definition).
ing to the general procedure for PFB derivatization. After that, they were analyzed by GC/MS.
3. Results and discussion 3.1. Derivatization by PFB-Tos
2.4. Detection of cyanide and azide in the beverages KCN was added to the coffee in order to become 1 mg/ml (38.4 mM) as a CN concentration. NaN3 was added to the cola in order to become 1 mg/ml (23.8 mM) as a N3 concentration. The water was removed in each sample under the nitrogen gas stream. Then, analysis was performed accord-
PFB derivatization was conducted with KF, NaCl, NaBr, NaI, KCN, KOCN, KSCN, NaN3, KNO3, NaNO2, KClO4, NaClO3, Na2SO4, Na2S2O3, and Na2CO3 by PFB-Tos. GC/MS analysis detected monovalent anions such as PFB-F, PFB-Cl, PFB-Br, PFB-I, PFB-CN, PFB-OCN, PFB-SCN, PFB-N3, PFB-NO3, and PFB-NO2, except for
Fig. 4. Mass spectra of pentafluorobenzyl inorganic anion derivatives. (A) Positive EI mass spectrum of PFB-SCN, (B) positive EI mass spectrum of PFB-N3, (C) positive EI mass spectrum of PFB-ONO2, (D) positive EI mass spectrum of PFB-ONO, (E) negative CI mass spectrum of PFB-ONO.
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Table 1 GC/MS results of inorganic anion derivatives Anion
Anion derivatized
Retention time (min)
Observed ions
Fluoride Chloride Bromide Iodide Cyanide Cyanate Thiocyanate Azide Nitrate Nitrite Nitritea
PFB-F PFB-Cl PFB-Br PFB-I PFB-CN PFB-OCN PFB-SCN PFB-N3 PFB-NO3 PFB-NO2 PFB-NO2
3.3 5.6 6.9 8.5 7.4 7.3 10.4 7.1 8.1 7.9 7.9
200 218 262 308 207 223 239 223 243 181 226
a
M+, 199 [M H]+, 181 [M F]+, 150 M+ (37Cl), 216 M+ (35Cl), 181 [M Cl]+, 161 M+ (81Br), 260 M+ (79Br), 181 [M Br]+, 161 M+, 196, 181 [M I]+, 161 M+, 188, 181 [M CN]+, 157 M+, 204, 193, 181 [M OCN]+ M+, 196, 181 [M SCN]+, 161 M+, 194 [M N2H]+, 181 [M N3]+ M+, 197, 195, 181 [M NO3]+, 167 [M NO2]+, 161 [M H] , 181 [M NO2] , 46 [NO2]
Figure Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
3A 3B 3C 3D 3E 3F 4A 4B 4C 4D 4E
CI negative
PFB-ClO4 and PFB-ClO3 were not detected; instead, PFBCl, which results from ClO4 and ClO3 , was detected. The ClO4 and ClO3 appeared to decompose with the evolution of O2 during the reaction. The PFB derivatives of bivalent anions; SO42 , S2O32 , and CO32 were not detected. It seems, SO42 would give only PFB-NaSO4 derivative so the reaction did not proceed to (PFB)2-SO4. 3.2. GC/MS of PFB derivatives GC/MS results for PFB derivatives of KF, NaCl, NaBr, NaI, KCN, KOCN, KSCN, NaN3, KNO3, and NaNO2 are shown as molecular ions (M+) of the derivatives in Fig. 2.
The mass spectra of the PFB derivatives of each inorganic anion are as shown in Figs. 3 and 4. Table 1 summarized the GC/MS result of inorganic anion derivatives concerning anion species, anion derivatized, retention time, and observed ions. The characteristic isotopical peaks of chloride and bromide were observed in the mass spectrum. A molecular ion peak of PFB derivative of nitrite (PFB-NO2) was not detected. Therefore, CI was performed to obtain molecular weight information. A molecular-related ion of m/ z 226 [M H] was detected in the CI (negative) spectrum. The PFB derivatives of azide and cyanate exhibit the same integral mass number of molecular weight 223. Fragment ions in the mass spectrum of the PFB derivative of cyanate are similar to those of the azide PFB derivative, although a
Fig. 5. Mass chromatogram of 15-crown-5, 18-crown-6, and PFB-Tos.
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peak at m/z 204 is detected for cyanate. Retention times of the two are also similar, preventing easy separation and identification. 3.3. Phase transfer catalyst Mass spectra of the phase transfer catalysts 15-Crown-5 and 18-Crown-6 are shown in Fig. 6A and B. Small peaks indicating molecular ions (M+) of 15-Crown-5 and 18Crown-6 occurred with retention times of 15.5 and 18.6 min, respectively, as shown in Fig. 5. In the PFB derivatization of inorganic anions, crown ether was used as a phase transfer catalyst instead of a
quaternary ammonium salt. Quaternary ammonium salts generally contain chloride or bromide ions, preventing the detection of Cl or Br ions in samples. The use of crown ethers eliminates this disadvantage. Derivatization of cyanate with PFB-Tos has been reported [12] using a quaternary ammonium salt as the phase transfer catalyst, but its PFB derivative was not detected. In this study, detection of the cyanate ion was possible using a crown ether as the phase transfer catalyst and PFB-Tos as the derivatization reagent. As sodium and potassium ions become incorporated in a cavity of the crown ether, the cyanate ion is liberated, increasing the reactivity of the cyanate ion. 3.4. Derivatization reagent The peak for PFB-Tos had a retention time of 19.0 min as shown in Fig. 5. The molecular ion at m/z 352 and fragment ion peaks at m/z 196, 181 [M Tos]+ and 156 were detected in the EI mass spectrum as shown in Fig. 6C. With PFB-Br as a derivatization reagent, retention times of PFB derivatives of inorganic anions were within 11 min and were close to that of PFB-Br (7 min), resulting in interference. However, the retention time of PFB-Tos is approximately 19 min, which differs greatly from PFB derivatives of inorganic anions. Therefore, the desired compounds can be detected without obstruction using PFB-Tos as a derivatization reagent. 3.5. Lower limit of the detection PFB derivatization of F , Cl , Br , I , CN , OCN , SCN , NO2 , NO3 , and N3 was conducted using PFBTos. The lower limit of the detection was determined by GC/MS. Concerning the detection limit, the concentration which gives full scan mass spectrum and can detect molecular ion, was defined as detection limit, because full scan mass spectrum is important in the forensic chemistry field as an evidential value. Results are shown in Table 2. Detection sensitivity of each derivative was less than 30 ng Table 2 Detection limits of inorganic anion derivatives
Fig. 6. Mass spectra of phase transfer catalyst and derivatizing reagent. (A) Positive EI mass spectrum of 15-crown-5, (B) positive EI mass spectrum of 18-crown-6, (C) positive EI mass spectrum of PFB-Tos.
Sample
Molar concentration (mM)
Amount of anions (ng)
Fluoride (F ) Chloride (Cl ) Bromide (Br ) Iodide (I ) Cyanide (CN ) Cyanate (OCN ) Thiocyanide (SCN ) Nitrite (NO2 )a Nitrate (NO3 ) Azide (N3 )
0.25 0.01 0.02 0.05 0.05 0.25 0.05 0.005 0.25 0.05
4.75 0.35 1.60 6.35 0.52 10.50 2.90 0.23 15.50 2.10
a
CI negative.
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Table 3 The accurate mass measurements of inorganic anion derivatives Compound
Molecular formula
Measured ion
Theoretical m/z
Measured mass m/z
Mass difference (mmu)
PFB-F PFB-Cl PFB-Br PFB-I PFB-CN PFB-OCN PFB-SCN PFB-ONO PFB-ONO2 PFB-N3
C7H2F6 C7H235Cl1F5 C7H279Br1F5 C7H2F5I1 C8H2N1F5 C8H2O1N1F5 C8H2S1N1F5 C7H1O2N1F5 C7H2O3N1F5 C7H2N3F5
M+ M+ M+ M+ M+ M+ M+ [M M+ M+
200.0061 215.9765 259.9260 307.9121 207.0107 223.0057 238.9828 225.9927 242.9955 223.0169
200.0056 215.9772 259.9260 307.9116 207.0113 223.0074 238.9817 225.9927 242.9964 223.0164
0.4 +0.7 +0.0 0.6 +0.6 +1.8 1.1 +0.0 +0.9 0.4
(EI) (EI) (EI) (EI) (EI) (EI) (EI) H] (CI) (EI) (EI)
approximately, which is sufficiently sensitive for practical analyses. PFB-NO2 was detectable within 5 pmol when the molecular-related ion [M H] was confirmed by the CI method, although the molecular ion was not detected by EI. 3.6. Accurate mass measurement PFB derivatization was conducted with F , Cl , Br , I , CN , OCN , SCN , NO2 , NO3 , and N3 using PFB-Tos. Accurate mass numbers for the M+ ion were obtained by EI+,
except for PFB-ONO. The M+ ion for PFB-ONO could not be detected using the EI+ method; therefore it was obtained by the CI (negative) method using the [M H] ion. Results are shown in Table 3. Experimental error was within 2 millimass units (mmu) of the actual mass of each PFB derivative. Consequently, the compositional formula could be obtained from the accurate mass number. The PFB derivatives of azide and cyanate exhibit the same integral mass number of molecular weight 223. Fragment ions and retention time of cyanate are similar to those
Fig. 7. GC/MS result of the derivatized obtained from coffee containing 38.4 mM of cyanide. (A) Mass chromatogram at m/z 207 (M+) of cyanide, (B) positive EI mass spectrum of PFB derivative of cyanide.
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Fig. 8. GC/MS result of the derivatized obtained from cola containing 23.8 mM of azide. (A) Mass chromatogram at m/z 223 (M+) of azide, (B) positive EI mass spectrum of PFB derivative of azide.
of the azide, preventing easy separation and identification. However, it is possible to determine the compositional formula of the desired compound by measuring an accurate mass number, allowing discrimination of the desired compound. In general, accurate mass measurements are obtained at a resolution of 3000 or 5000 (10% valley definition) and perfluorokerosen (PFK) is used as a mass calibration compound. Accurate mass measurements of PFB derivatives of inorganic anions were obtained at a resolution of 3000 (10% valley definition), and corresponding elemental compositions were within an experimental error of 2 mmu. The theoretical mass number of PFB-ONO2 is m/z 242.9955. One PFK peak occurs at m/z 242.9856 (C6F9), which is a difference of 9.9 mmu. A resolution of 24,544 is necessary to separate these two compounds [resolution = m1/(m2 m1)]. However, the mass spectrometer used for this study was unable to achieve this level of resolution. Therefore, PFK was introduced into the ion source immediately after the sample peak was obtained. In conclusion, accurate mass measurements were obtained with a resolution of 3000 by separately introducing the sample and PFK into the ion source.
3.7. Detection of cyanide and azide in the beverages As a result of measuring the cyanide in the coffee, mass chromatograph showed the peak at m/z 207 of molecular ion of cyanide (Fig. 7A), and the mass spectrum of this peak (Fig. 7B) was the same as that of the PFB-CN (Fig. 3E). As a result of measuring the azide in the cola, mass chromatograph showed the peak at m/z 223 of molecular ion of azide (Fig. 8A), and the mass spectrum (Fig. 8B) of this peak was the same as that of the PFB-N3 (Fig. 4B). It was possible to detect the inorganic anion from the beverages of coffee and cola. This method is regarded applied possibly not only in forensic science but also food chemistry and environmental science.
4. Conclusion A sensitive, simple and systematic method for analyzing inorganic anions by GC/MS was devised for derivatization by using of PFB-Tos as derivatizing regent, and crown ether as a phase transfer catalyst. This method could detected F, Cl, Br, I, CN, OCN, SCN, N3, NO3, and NO2. Accurate mass
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number of PFB derivatives could give the compositional formula which make it possible to identify the inorganic anions having the same integral mass number. In addition, this method was applied to the actual trial sample of matrix.
Acknowledgements Parts of this work were supported by grants-in-aid for Science and Research (B) from the Japan Society for the Promotion of Science to MS (grant no. 15915014).
[4] [5] [6] [7] [8] [9] [10] [11] [12]
References [1] H. Small, T.S. Stevens, W.C. Bauman, Anal. Chem. 47 (1975) 1801–1809. [2] P.R. Haddad, J. Chromatogr. A 770 (1997) 281–290. [3] J. Romano, P. Jandik, W.R. Jones, P.E. Jackson, J. Chromatogr. 546 (1991) 411–421.
[13] [14] [15] [16]
143
P. Jandik, W.R. Jones, J. Chromatogr. 546 (1991) 431–443. W.R. Jones, J. Chromatogr. 640 (1993) 387–395. T. Soga, G.A. Ross, J. Chromatogr. A 767 (1997) 223–230. T. Soga, G.A. Ross, J. Chromatogr. A 834 (1999) 65–71. T. Soga, I. Tajima, D.N. Heiger, Am. Lab. (2000) 124– 126. K. Funazo, M. Tanaka, T. Shono, J. Chromatogr. 211 (1981) 361–368. M. Tanaka, K. Funazo, T. Hirashima, T. Shono, J. Chromatogr. 234 (1982) 373–379. K. Funazo, H.L. Wu, K. Morita, M. Tanaka, T. Shono, J. Chromatogr. 319 (1985) 143–152. K. Funazo, M. Tanaka, K. Morita, M. Kamino, T. Shono, J. Chromatogr. 346 (1985) 215–225. S. Kage, T. Nagata, K. Kudo, J. Chromatogr. B 675 (1996) 27– 32. S. Kage, K. Kudo, N. Ikeda, J. Anal. Toxicol. 26 (2002) 320– 324. S. Kage, K. Kudo, N. Ikeda, J. Anal. Toxicol. 24 (2000) 429– 432. K. Tsuge, Y. Seto, Jpn. J. Sci. Tech. Iden. 7 (2002) 19–35.
Identification of inorganic anions by gas chromatography/mass spectrometry Masataka Sakayanagi a,*, Yaeko Yamada b, Chikako Sakabe b, Kunio Watanabe a, Yoshihiro Harigaya b a
Scientific Criminal Investigation Laboratory, Kanagawa Prefectural Police Headquarters, 155-1 Yamashita-Cho, Naka-Ku, Yokohama 231-0023, Japan b School of Pharmaceutical Science, Kitasato University, 5-9-1 Shirokane Minato-ku, Tokyo 108-8641, Japan Received 25 February 2005; accepted 8 April 2005 Available online 10 May 2005
Abstract Inorganic anions were identified by using gas chromatography/mass spectrometry (GC/MS). Derivatization of the anions was achieved with pentafluorobenzyl p-toluenesulphonate (PFB-Tos) as the reaction reagent and a crown ether as a phase transfer catalyst. When PFB-Br was used as the reaction regent, the retention time of it was close to those of the derivatized inorganic anions and interfered with the analysis. In contrast, the retention time of PFB-Tos differed greatly from the PFB derivatives of the inorganic anions and the compounds of interest could be detected without interference. Although the PFB derivatives of SO4, S2O3, CO3, ClO4, and ClO3 could not be detected, the derivatives of F, Cl, Br, I, CN, OCN, SCN, N3, NO3, and NO2 were detected using PFB-Tos as the derivatizing reagent. The inorganic anions were detectable within 30 ng approximately, which is of sufficient sensitivity for use in forensic chemistry. Accurate mass number was measured for each PFB derivative by high-resolution mass spectrometry (HRMS) within a measurement error of 2 millimass units (mmu), which allowed determination of the compositional formula from the mass number. In addition, actual analysis was performed successively by our method using trial samples of matrix. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic science; Inorganic anion; Pentafluorobenzyl derivatives; Crown ether; Gas chromatography; Mass spectrometry
1. Introduction The analysis of inorganic anions in forensic chemistry is important for the analysis of powder residue from explosives and contamination of poisons. Generally, analysis of inorganic anions is accomplished by ion chromatography [1,2]. Recently, capillary electrophoresis methods [2–8] have been developed.
* Corresponding author. Tel.: +81 45 662 0395; fax: +81 45 662 0395. E-mail address: [email protected] (M. Sakayanagi).
Ion chromatography and capillary electrophoresis identify inorganic anions by retention time or migration time. However, a sample contaminated with matrix is often difficult to analyze. Gas chromatography/mass spectrometry (GC/MS) identifies compounds by chromatography retention time and mass spectrum, making the method a highly accurate procedure and an essential method in forensic chemistry. Inorganic anions are not volatile, so analysis by gas chromatography directly is difficult. Therefore, GC methods for inorganic ions require conversion to a volatile derivative such as a methyl [9], ethyl [10], butyl [11], or pentafluorobenzyl (PFB) derivative [12–16]. Among them, PFB derivatives have been used for supersensitive ECD
0379-0738/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2005.04.003
M. Sakayanagi et al. / Forensic Science International 157 (2006) 134–143
(electron capture detector) in GC [12] and GC/MS [13–16]. The conventional synthesis of PFB derivatives of inorganic anions uses a quaternary ammonium salt as a phase transfer catalyst, with the reaction conducted between an organic (salt of anion) and an aqueous (PFB-Br and PFB derivative of anion) phase. Funazo et al. [12] reported that bromide, iodide, cyanide, thiocyanate, nitrite, nitrate, and sulfide could be converted to PFB derivatives using pentafluorobenzyl p-toluenesulphonate (PFB-Tos) as the derivatizing reagent and tetra-n-amylammonium chloride as a phase transfer catalyst. The PFB derivatives were analyzed by GC with a flame ionization detector. But this method was not ideal for the identification of unknown ions, because GC discriminated among unknown agents only by retention time. Kage et al. reported the analysis of PFB derivatives of inorganic anions by GC/MS. Cyanide [13], thiocyanate [13], nitrite [14], nitrate [14], and azide [15] were converted to PFB derivatives using pentafluorobenzyl bromide (PFB-Br) and tetra-decyldimethylbenzylammonium chloride (TDMBA) as a phase transfer catalyst. In addition, Tsuge et al. [16] reported that cyanide, thiocyanate, nitrite, nitrate, azide, and sulfide could be converted to PFB derivatives using PFB-Br as the derivatizing reagent and TDMBA or polymer-bound tributylmethylphoshonium chloride as a phase transfer catalyst, and then identified by GC/MS. However, when the quaternary ammonium salt was used as the phase transfer catalyst as shown in the case of Kage et al. and Tsuge et al., the anion of the salt (e.g., chloride or bromide ion) was present in the reaction mixture. Therefore, the detection of chloride or bromide was difficult. This study was designed to develop a method for the discrimination of inorganic anions under a single condition. Crown ethers were used as a phase transfer catalyst because they do not contain inorganic anions. The inorganic anions were derivatized between a solid (salt of anion) and liquid (PFB-Tos and PFB derivative of anion) phase by PFB-Tos, using a crown-ether as the phase transfer catalyst. This resulted in a more convenient reaction process than the previously reported processes. PFB derivatives were detected by GC/MS, and detection limits were determined. Furthermore, the application of our method to trial samples of matrix was actually performed by using beverages which contained azide and cyanide; because, sodium
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azide and potassium cyanide become a problem in matters relating to poisoning in Japan. For low-resolution mass spectrometry (LRMS), the same mass number can indicate different compositions. Because LRMS determines integral mass; it cannot unambiguously identify a compound. Then, accurate mass measurement by high-resolution mass spectrometry (HRMS) becomes important. Accurate mass measurement of PFB derivatives of inorganic anions to identify compositional formula using highresolution GC/MS has not yet been reported. Therefore, we determined the compositional formula of PFB derivatives of inorganic anions by measurement of the mass number.
2. Experimental 2.1. Materials 2.1.1. Inorganic anions Inorganic anions were purchased from Wako Pure Chemical Industries Co. Ltd. (Osaka, Japan) and Kanto Chemistry Co. Ltd. (Tokyo, Japan). The following reagents were used: potassium fluoride (KF), sodium chloride (NaCl), sodium bromide (NaBr), sodium iodide (NaI), potassium cyanide (KCN), potassium cyanate (KOCN), potassium thiocyanate (KSCN), sodium azide (NaN3), potassium nitrate (KNO3), sodium nitrite (NaNO2), potassium perchlorate (KClO4), sodium chlorate (NaClO3), sodium sulfate (Na2SO4), sodium thiosulfate (Na2S2O3), and sodium carbonate (Na2CO3). 2.1.2. Derivatizing reagent Pentafluorobenzyl p-toluene sulfonate (PFB-Tos, TCI; Tokyo, Japan) was used for the derivatizing regent. PFB-Tos (1.761 g) was dissolved in 100 ml of dichloromethane to give a concentration of 50 mM. 2.1.3. Phase transfer catalysts The 18-crown-6-ether (18-crown-6, Wako Pure Chemical Industries Co. Ltd., Osaka, Japan) and 15-crown-5-ether (15-crown-5, Aldrich; St. Louis, MO, USA) were used as phase transfer catalysts. 18-crown-6 (264.3 mg) and 15crown-5 (220.2 mg) were dissolved in 100 ml of dichloromethane to give a concentration of 10 mM.
Fig. 1. Formula for the PFB derivatives.
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The salts of inorganic ions are usually insoluble in organic solvents. However, cations such as sodium and potassium become trapped in the cavity of some crown ethers that act as catalysts. A segregated anion with high reactivity is formed
with the crown ether and becomes soluble in organic solvents. 15-crown-5 chelates sodium ions, while 18-crown-6 chelates potassium and ammonium ions. The other regents used were of analytical grade.
Fig. 2. Mass chromatograms of pentafluorobenzyl inorganic anion derivatives.
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2.2. General procedure for PFB derivatization A 0.01 mmol inorganic anion, 0.2 ml volume of 50 mM PFB-Tos solution, 0.1 ml of 10 mM crown ether solution, and 0.9 ml of dichloromethane were put into a 5 ml volume of glass vial for derivatization. After the mixture was stirred at room temperature for 90 min, it was diluted with dichloromethane, and injected into the GC/MS. Fig. 1 outlines the PFB derivatization of inorganic anions. 2.3. GC/MS conditions GC/MS analysis was performed with a Hewlett-Packard HP-5890II GC instrument (Palo Alto, CA, USA) equipped
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with a JEOL JMS-AX505HA double-focusing mass spectrometer (Tokyo, Japan). The GC was equipped with an HP-1MS-fused silica capillary column (30 m 0.25 mm inside diameter, 0.25 mm film thickness). Temperature was programmed from 45 8C (1.5 min hold) to 280 8C at 10 8C/ min. Helium was used as the carrier gas. The injection port was operated in splitless mode and valve off time was set at 1 min. GC injection port and transfer line temperatures were 250 and 300 8C, respectively. GC injection port pressure was 11 psi. Ionization energy of positive-ion electron impact ionization (EI) was 70 eV. Ionization energy and the reagent gas of the negative-ion chemical ionization (CI) conditions were 200 eVand isobutene, respectively. The ionizing current was measured at 300 mA. HRMS was
Fig. 3. Mass spectra of pentafluorobenzyl inorganic anion derivatives. (A) Positive EI mass spectrum of PFB-F, (B) positive EI mass spectrum of PFB-Cl, (C) positive EI mass spectrum of PFB-Br, (D) positive EI mass spectrum of PFB-I, (E) positive EI mass spectrum of PFB-CN, (F) positive EI mass spectrum of PFB-OCN.
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conducted by the electric field scanning method, and LRMS was conducted by the magnetic field scanning method. HRMS experiments were performed at a resolution of 3000 (10% valley definition), and LRMS experiments were performed at a resolution of 500 (10% valley definition).
ing to the general procedure for PFB derivatization. After that, they were analyzed by GC/MS.
3. Results and discussion 3.1. Derivatization by PFB-Tos
2.4. Detection of cyanide and azide in the beverages KCN was added to the coffee in order to become 1 mg/ml (38.4 mM) as a CN concentration. NaN3 was added to the cola in order to become 1 mg/ml (23.8 mM) as a N3 concentration. The water was removed in each sample under the nitrogen gas stream. Then, analysis was performed accord-
PFB derivatization was conducted with KF, NaCl, NaBr, NaI, KCN, KOCN, KSCN, NaN3, KNO3, NaNO2, KClO4, NaClO3, Na2SO4, Na2S2O3, and Na2CO3 by PFB-Tos. GC/MS analysis detected monovalent anions such as PFB-F, PFB-Cl, PFB-Br, PFB-I, PFB-CN, PFB-OCN, PFB-SCN, PFB-N3, PFB-NO3, and PFB-NO2, except for
Fig. 4. Mass spectra of pentafluorobenzyl inorganic anion derivatives. (A) Positive EI mass spectrum of PFB-SCN, (B) positive EI mass spectrum of PFB-N3, (C) positive EI mass spectrum of PFB-ONO2, (D) positive EI mass spectrum of PFB-ONO, (E) negative CI mass spectrum of PFB-ONO.
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Table 1 GC/MS results of inorganic anion derivatives Anion
Anion derivatized
Retention time (min)
Observed ions
Fluoride Chloride Bromide Iodide Cyanide Cyanate Thiocyanate Azide Nitrate Nitrite Nitritea
PFB-F PFB-Cl PFB-Br PFB-I PFB-CN PFB-OCN PFB-SCN PFB-N3 PFB-NO3 PFB-NO2 PFB-NO2
3.3 5.6 6.9 8.5 7.4 7.3 10.4 7.1 8.1 7.9 7.9
200 218 262 308 207 223 239 223 243 181 226
a
M+, 199 [M H]+, 181 [M F]+, 150 M+ (37Cl), 216 M+ (35Cl), 181 [M Cl]+, 161 M+ (81Br), 260 M+ (79Br), 181 [M Br]+, 161 M+, 196, 181 [M I]+, 161 M+, 188, 181 [M CN]+, 157 M+, 204, 193, 181 [M OCN]+ M+, 196, 181 [M SCN]+, 161 M+, 194 [M N2H]+, 181 [M N3]+ M+, 197, 195, 181 [M NO3]+, 167 [M NO2]+, 161 [M H] , 181 [M NO2] , 46 [NO2]
Figure Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
3A 3B 3C 3D 3E 3F 4A 4B 4C 4D 4E
CI negative
PFB-ClO4 and PFB-ClO3 were not detected; instead, PFBCl, which results from ClO4 and ClO3 , was detected. The ClO4 and ClO3 appeared to decompose with the evolution of O2 during the reaction. The PFB derivatives of bivalent anions; SO42 , S2O32 , and CO32 were not detected. It seems, SO42 would give only PFB-NaSO4 derivative so the reaction did not proceed to (PFB)2-SO4. 3.2. GC/MS of PFB derivatives GC/MS results for PFB derivatives of KF, NaCl, NaBr, NaI, KCN, KOCN, KSCN, NaN3, KNO3, and NaNO2 are shown as molecular ions (M+) of the derivatives in Fig. 2.
The mass spectra of the PFB derivatives of each inorganic anion are as shown in Figs. 3 and 4. Table 1 summarized the GC/MS result of inorganic anion derivatives concerning anion species, anion derivatized, retention time, and observed ions. The characteristic isotopical peaks of chloride and bromide were observed in the mass spectrum. A molecular ion peak of PFB derivative of nitrite (PFB-NO2) was not detected. Therefore, CI was performed to obtain molecular weight information. A molecular-related ion of m/ z 226 [M H] was detected in the CI (negative) spectrum. The PFB derivatives of azide and cyanate exhibit the same integral mass number of molecular weight 223. Fragment ions in the mass spectrum of the PFB derivative of cyanate are similar to those of the azide PFB derivative, although a
Fig. 5. Mass chromatogram of 15-crown-5, 18-crown-6, and PFB-Tos.
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peak at m/z 204 is detected for cyanate. Retention times of the two are also similar, preventing easy separation and identification. 3.3. Phase transfer catalyst Mass spectra of the phase transfer catalysts 15-Crown-5 and 18-Crown-6 are shown in Fig. 6A and B. Small peaks indicating molecular ions (M+) of 15-Crown-5 and 18Crown-6 occurred with retention times of 15.5 and 18.6 min, respectively, as shown in Fig. 5. In the PFB derivatization of inorganic anions, crown ether was used as a phase transfer catalyst instead of a
quaternary ammonium salt. Quaternary ammonium salts generally contain chloride or bromide ions, preventing the detection of Cl or Br ions in samples. The use of crown ethers eliminates this disadvantage. Derivatization of cyanate with PFB-Tos has been reported [12] using a quaternary ammonium salt as the phase transfer catalyst, but its PFB derivative was not detected. In this study, detection of the cyanate ion was possible using a crown ether as the phase transfer catalyst and PFB-Tos as the derivatization reagent. As sodium and potassium ions become incorporated in a cavity of the crown ether, the cyanate ion is liberated, increasing the reactivity of the cyanate ion. 3.4. Derivatization reagent The peak for PFB-Tos had a retention time of 19.0 min as shown in Fig. 5. The molecular ion at m/z 352 and fragment ion peaks at m/z 196, 181 [M Tos]+ and 156 were detected in the EI mass spectrum as shown in Fig. 6C. With PFB-Br as a derivatization reagent, retention times of PFB derivatives of inorganic anions were within 11 min and were close to that of PFB-Br (7 min), resulting in interference. However, the retention time of PFB-Tos is approximately 19 min, which differs greatly from PFB derivatives of inorganic anions. Therefore, the desired compounds can be detected without obstruction using PFB-Tos as a derivatization reagent. 3.5. Lower limit of the detection PFB derivatization of F , Cl , Br , I , CN , OCN , SCN , NO2 , NO3 , and N3 was conducted using PFBTos. The lower limit of the detection was determined by GC/MS. Concerning the detection limit, the concentration which gives full scan mass spectrum and can detect molecular ion, was defined as detection limit, because full scan mass spectrum is important in the forensic chemistry field as an evidential value. Results are shown in Table 2. Detection sensitivity of each derivative was less than 30 ng Table 2 Detection limits of inorganic anion derivatives
Fig. 6. Mass spectra of phase transfer catalyst and derivatizing reagent. (A) Positive EI mass spectrum of 15-crown-5, (B) positive EI mass spectrum of 18-crown-6, (C) positive EI mass spectrum of PFB-Tos.
Sample
Molar concentration (mM)
Amount of anions (ng)
Fluoride (F ) Chloride (Cl ) Bromide (Br ) Iodide (I ) Cyanide (CN ) Cyanate (OCN ) Thiocyanide (SCN ) Nitrite (NO2 )a Nitrate (NO3 ) Azide (N3 )
0.25 0.01 0.02 0.05 0.05 0.25 0.05 0.005 0.25 0.05
4.75 0.35 1.60 6.35 0.52 10.50 2.90 0.23 15.50 2.10
a
CI negative.
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Table 3 The accurate mass measurements of inorganic anion derivatives Compound
Molecular formula
Measured ion
Theoretical m/z
Measured mass m/z
Mass difference (mmu)
PFB-F PFB-Cl PFB-Br PFB-I PFB-CN PFB-OCN PFB-SCN PFB-ONO PFB-ONO2 PFB-N3
C7H2F6 C7H235Cl1F5 C7H279Br1F5 C7H2F5I1 C8H2N1F5 C8H2O1N1F5 C8H2S1N1F5 C7H1O2N1F5 C7H2O3N1F5 C7H2N3F5
M+ M+ M+ M+ M+ M+ M+ [M M+ M+
200.0061 215.9765 259.9260 307.9121 207.0107 223.0057 238.9828 225.9927 242.9955 223.0169
200.0056 215.9772 259.9260 307.9116 207.0113 223.0074 238.9817 225.9927 242.9964 223.0164
0.4 +0.7 +0.0 0.6 +0.6 +1.8 1.1 +0.0 +0.9 0.4
(EI) (EI) (EI) (EI) (EI) (EI) (EI) H] (CI) (EI) (EI)
approximately, which is sufficiently sensitive for practical analyses. PFB-NO2 was detectable within 5 pmol when the molecular-related ion [M H] was confirmed by the CI method, although the molecular ion was not detected by EI. 3.6. Accurate mass measurement PFB derivatization was conducted with F , Cl , Br , I , CN , OCN , SCN , NO2 , NO3 , and N3 using PFB-Tos. Accurate mass numbers for the M+ ion were obtained by EI+,
except for PFB-ONO. The M+ ion for PFB-ONO could not be detected using the EI+ method; therefore it was obtained by the CI (negative) method using the [M H] ion. Results are shown in Table 3. Experimental error was within 2 millimass units (mmu) of the actual mass of each PFB derivative. Consequently, the compositional formula could be obtained from the accurate mass number. The PFB derivatives of azide and cyanate exhibit the same integral mass number of molecular weight 223. Fragment ions and retention time of cyanate are similar to those
Fig. 7. GC/MS result of the derivatized obtained from coffee containing 38.4 mM of cyanide. (A) Mass chromatogram at m/z 207 (M+) of cyanide, (B) positive EI mass spectrum of PFB derivative of cyanide.
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Fig. 8. GC/MS result of the derivatized obtained from cola containing 23.8 mM of azide. (A) Mass chromatogram at m/z 223 (M+) of azide, (B) positive EI mass spectrum of PFB derivative of azide.
of the azide, preventing easy separation and identification. However, it is possible to determine the compositional formula of the desired compound by measuring an accurate mass number, allowing discrimination of the desired compound. In general, accurate mass measurements are obtained at a resolution of 3000 or 5000 (10% valley definition) and perfluorokerosen (PFK) is used as a mass calibration compound. Accurate mass measurements of PFB derivatives of inorganic anions were obtained at a resolution of 3000 (10% valley definition), and corresponding elemental compositions were within an experimental error of 2 mmu. The theoretical mass number of PFB-ONO2 is m/z 242.9955. One PFK peak occurs at m/z 242.9856 (C6F9), which is a difference of 9.9 mmu. A resolution of 24,544 is necessary to separate these two compounds [resolution = m1/(m2 m1)]. However, the mass spectrometer used for this study was unable to achieve this level of resolution. Therefore, PFK was introduced into the ion source immediately after the sample peak was obtained. In conclusion, accurate mass measurements were obtained with a resolution of 3000 by separately introducing the sample and PFK into the ion source.
3.7. Detection of cyanide and azide in the beverages As a result of measuring the cyanide in the coffee, mass chromatograph showed the peak at m/z 207 of molecular ion of cyanide (Fig. 7A), and the mass spectrum of this peak (Fig. 7B) was the same as that of the PFB-CN (Fig. 3E). As a result of measuring the azide in the cola, mass chromatograph showed the peak at m/z 223 of molecular ion of azide (Fig. 8A), and the mass spectrum (Fig. 8B) of this peak was the same as that of the PFB-N3 (Fig. 4B). It was possible to detect the inorganic anion from the beverages of coffee and cola. This method is regarded applied possibly not only in forensic science but also food chemistry and environmental science.
4. Conclusion A sensitive, simple and systematic method for analyzing inorganic anions by GC/MS was devised for derivatization by using of PFB-Tos as derivatizing regent, and crown ether as a phase transfer catalyst. This method could detected F, Cl, Br, I, CN, OCN, SCN, N3, NO3, and NO2. Accurate mass
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number of PFB derivatives could give the compositional formula which make it possible to identify the inorganic anions having the same integral mass number. In addition, this method was applied to the actual trial sample of matrix.
Acknowledgements Parts of this work were supported by grants-in-aid for Science and Research (B) from the Japan Society for the Promotion of Science to MS (grant no. 15915014).
[4] [5] [6] [7] [8] [9] [10] [11] [12]
References [1] H. Small, T.S. Stevens, W.C. Bauman, Anal. Chem. 47 (1975) 1801–1809. [2] P.R. Haddad, J. Chromatogr. A 770 (1997) 281–290. [3] J. Romano, P. Jandik, W.R. Jones, P.E. Jackson, J. Chromatogr. 546 (1991) 411–421.
[13] [14] [15] [16]
143
P. Jandik, W.R. Jones, J. Chromatogr. 546 (1991) 431–443. W.R. Jones, J. Chromatogr. 640 (1993) 387–395. T. Soga, G.A. Ross, J. Chromatogr. A 767 (1997) 223–230. T. Soga, G.A. Ross, J. Chromatogr. A 834 (1999) 65–71. T. Soga, I. Tajima, D.N. Heiger, Am. Lab. (2000) 124– 126. K. Funazo, M. Tanaka, T. Shono, J. Chromatogr. 211 (1981) 361–368. M. Tanaka, K. Funazo, T. Hirashima, T. Shono, J. Chromatogr. 234 (1982) 373–379. K. Funazo, H.L. Wu, K. Morita, M. Tanaka, T. Shono, J. Chromatogr. 319 (1985) 143–152. K. Funazo, M. Tanaka, K. Morita, M. Kamino, T. Shono, J. Chromatogr. 346 (1985) 215–225. S. Kage, T. Nagata, K. Kudo, J. Chromatogr. B 675 (1996) 27– 32. S. Kage, K. Kudo, N. Ikeda, J. Anal. Toxicol. 26 (2002) 320– 324. S. Kage, K. Kudo, N. Ikeda, J. Anal. Toxicol. 24 (2000) 429– 432. K. Tsuge, Y. Seto, Jpn. J. Sci. Tech. Iden. 7 (2002) 19–35.