2647
Anal. Chem. 1887, 59, 2647-2652
Determination of Sulfonated Azo Dyes by Liquid Chromatography/Atmospheric Pressure Ionization Mass Spectrometry Andries P. Bruins,' Lars 0. G. Weidolf? and Jack D. Henion*
Drug Testing and Toxicology, N Y S College of Veterinary Medicine, Cornel1 University, Ithaca, New York 14850 William L. Budde
Environmental Monitoring and Support Laboratory, Office of Research and Development, US.Environmental Protection Agency, Cincinnati, Ohio 45268
Sulfonated azo dyes are separated by highperformance ilquid chromatography and introduced into the atmospherk pressure ion source of a trlple quadrupole mass spectrometer uslng pneumatic nebulizers. A heated nebulizer together wlth a corona dlscharge produces ions by gas phase chemical lonIzath. Two related nebulizers operating at room temperature and fioatlng at a high vonage make use of Ions present In the liquid phase. Collision induced dissociation of [M HI- ions shows SOs'- as a fragment characteristic of sulfonated azo dyes and parent ion scans on SO3*- are used for identlflcation of azo dyes In environmental samples. Selected ion monitoring and selected reaction monltorlng are used for the determination of speciflc dyes.
-
The chemistry of dyes from natural resources has a long history going back to the dye industry of the Phoenicians in the 13th Century B.C. ( I ) . The manufacturing of azo dyes was one of the cornerstones of the coal tar based chemical industry, while the development of synthetic dyes was intimately related to the evolution of organic chemistry in the late 19th century. Each year millions of kilograms of azo dyes are produced and used in diverse applications including textile dyes, paint pigments, printing inks, and food coloring. During the manufacture and use of azo dyes, wastes are created and subsequently discarded as wastewater and solid residues. In recent years a number of azo dyes have been removed from the list of substances approved for use in foods, drugs, and cosmetics, and a number of others are under study with regard to potential adverse health effects. In addition, azo dyes can be reduced in the environment (2) and in vivo (3) to produce amines that are known carcinogens. Therefore the presence of azo dyes in wastewater and solid waste is of considerable interest because of the potential for contamination of groundwater and drinking water supplies with compounds that may produce health risks. Azo dyes have the general structure I, where Ar and Ar' are substituted aromatic systems, usually phenyl or naphthyl. "he aromatic ring systems are invariably substituted by one or more of a large variety of electron-donating and electronwithdrawing groups. The substituents often contain additional azo groups, chelated metal ions, and charged organic functional groups. The wide variety of structures and highly delocalized electron distributions account for the great variety of colored dyes and pigments in the azo dye series. On temporary leave from State University, Department of Pharmacy, A. Deusinglaan 2,9713AW Groningen, The Netherlands. *On temporary leave from AB Hiissle, S-43183Molndal, Sweden. 0003-2700/87/0359-2647$01.50/0
Ar-N=N-Ar' I Published analytical methods for azo dyes are frequently based on the ultraviolet-visible (UV-Vis) spectra of these compounds (4). For many sample types, including wastewater and solid waste, separation of the components in complex mixtures is required before measurement, and separations using high-performance liquid chromatography (HPLC) have been reported (5,6). Unfortunately, the potential for interferences and errors in HPLC/UV-Vis methods is high since the resolution obtained by HPLC is not always sufficient to completely resolve all components in complex mixtures, and a great number of naturally occurring and industrial compounds also absorb in the UV-Vis region. Therefore, in order to assess the potential environmental impact of this series of compounds, definitive analytical methods are needed. A method based on some other spectroscopic property to allow for the independent identification and confirmation of the presence of a specific azo dye compound or compounds in environmental samples would be highly desirable. Structure elucidation or identification of dyes by mass spectrometry has been reported by using electron ionization for nonpolar petroleum azo dyes (7)and field desorption (8) or fast atom bombardment (9) for very polar colorants such as sulfonated and phosphonated azo dyes. The technique of liquid chromatography/mass spectrometry (LC/MS) has developed rapidly in recent years and has the potential to provide a needed definitive method. In work reported earlier, which was a part of this exploratory research program, Voyksner (10) found that certain azo dyes were amenable to analyses by LC/MS using the thermospray interface with either ammonium acetate or 200-eV filament electrons as the ionizing agent. Azo dyes gave intense [M + 1]+ions when the aromatic rings contained substituents such as hydroxy, methoxy, alkyl, acetamido, nitro, and dialkylamino. Betowski and Ballard have demonstrated the feasibility of thermospray for LC/MS/MS of some basic indolium cationic organic dyes (11). On the other hand, azo dyes containing sulfonic acid or sulfonate groups (acid azo dyes) did not produce any discernible positive or negative ions with the two modes of ionization under the LC conditions used (12). Since acid azo dyes are among the most important and widely used, a project was started to seek an alternative LC/MS technique for this group of azo dyes. Atmospheric pressure ionization (API) sources are compatible with liquid chromatography and offer a number of distinct advantages for LC/MS analyses. These ion sources are not limited by vacuum system constraints common in other types of systems. Simple pneumatic nebulizers have been developed (13) to interface liquid chromatography effluents to API sources and allow either gas-phase ionization 0 1987 American Chemical Society
2848
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
Chart I. Mono- and Disulfonated Azo Dyes R C I O REO 337 R C I D RED 1
R C I O RED I 5 1
8% No
No038 nu*sea
R C I O BLRCU 52 ,OH HO,
A C I D RED 88 M I X OF 2 ISOMERS HO
A C I D BLRCK 1
CDMPLEXED U I T H CHROMIUn
R C I D BLUE 1 1 8 R C I D VELLOU 1 5 1
A C I D ORANGE 7
H2NOgS
nu
9
87s
numaso
COMPLEXED U I T H COBALT
by corona discharge or liquid-phase ionization in an electric field (14). These API techniques appeared to have the potential of producing the ions needed for a successful LC/MS method for the determination of acid azo dyes. It was discovered during the course of this work that both modes of ionization generated ions amenable to mass spectrometry with mono- and disulfonated azo dyes and with a nonionic azo dye having a sulfonamide function and complexed with cobalt. In addition, a new "ion spray" LC/MS interface was developed from this work which does not utilize supplemented heat and demonstrates approximately 10-50 ng full-scan sensitivity for both mono- and disulfonated azo dyes in this study.
EXPERIMENTAL SECTION Reagents. The structures of the mono- and disulfonated dyes utilized in this study are given in Chart I. These dyes were obtained from the Ecological and Toxicological Association of the Dye Stuffs Manufacturing Industry. Organic solvents used for HPLC were obtained from Fisher Scientific (Rochester, NY)and HPLC grade water was obtained from a water purification system (Barnstead, Nanopure 11, Boston, MA). The mobile phases for HPLC were sonicated, sparged with He, and filtered through 0.45-pm Nylon-66 filters (Rainin, Woburn, MA) before use. Liquid Chromatography. Two dual pump HPLC systems (Waters, Milford, MA) were used throughout the study: two M6000A pumps controlled by an M660 solvent programmer, an M440 UV detector at 254 nm, and a Rheodyne (Cotati, CA) Model 7010 injection valve fitted with a 20-pL loop; or two M510 pumps, an M680 automated gradient controller, a constant temperature column oven, an M440 UV detector at 254 nm, and a Rheodyne Model 7125 injection valve equipped with a 20-wL loop. Microbore LC was performed with a single M510 solvent delivery system, a Rheodyne Model 7520 injection valve equipped with 0.2- or 1.0-pL loops and a 1.8-pLflow-through cell installed in the M440 UV detector. Sample Preparation. Solid Phase Extraction and Recovery of Dyes from Cayuga Lake Water. The monosulfonated dyes were extracted with disposable CIScartridges (J.T. Baker, Phillipsbmg, NJ, 3 mL, 200 mg, Model 7020-2) with a'vacuum manifold (Supelco, Bellefonte, PA). The columns were preconditioned with 2 mL of methanol followed by 2 mL of 0.01% triethylamine (TEA) in water. (It was necessary in include TEA in all solutions to
prevent irreversible adsorption of the dyes presumably to residual silanol groups on the bonded silica surface.) The sample applied was 100 pL of a standard dye mixture (acid orange 7, acid red 337, acid red 88 (two isomers), and acid red 151, approximately 10 pg of each dye) added to 100 mL of filtered Cayuga Lake water to which 10 pL of TEA had been added. Following extraction, the columns were washed with 2 mL of 0.01% TEA in water and 1 mL of 25% methanol in 0.01% TEA/water. The dyes were eluted with 2 mL of 85% methanol in 0.01% TEA/water. The eluate was evaporated at 60 "C under a stream of nitrogen and reconstituted in 100 pL of 25% acetonitrile in water. Recovery was determined by microbore LC using a Shandon 1 mm i.d. X 100 mm Hypersil3-pm CIS(Sewickley, PA) column with 30% acetonitrile/O.l M ammonium acetate as the mobile phase at a flow rate of 40 pL/min. The ratio of the peak areas obtained for the processed samples over the peak areas of the directly injected dye mixture provided recoveries of acid orange 7 , acid red 337, acid red 88 (two isomers), and acid red 151 which were 103%, 102%, 101%, 95%, and 87%, respectively. The solid phase extraction procedure was modified for the disulfonated dyes (acid red 1, acid black 1, and acid blue 113) since the highly polar acid red 1was only slightly retained by the bonded silica; a larger CIScartridge was used (J. T. Baker, 6 mL 1g, Model 7020-7) and washing of the column with 25% methanol in 0.01% TEA/water was omitted. The recoveries were determined by gradient elution HPLC with a Perkin-Elmer (Norwalk, CT) Pecosphere 3x3 CIS4.6 mm i.d. X 30 mm 3-pm column with a linear gradient of 25% B t o 100% B in 3 min at 1.0 mL/min, with mobile phases A and B being 10% and 90% methanol in 0.024 M ammonium acetate at pH 5.5, respectively. The recoveries of acid red 1,acid black 1,and acid blue 113 were loo%, 106%, and 65 % , respectively. Solid-Phase Extraction and Liquid Chromatography Screening
The wastewater samples were obtained from The Water Engineering Research Laboratory (Cincinnati, OH). The samples (100 mL including 0.01% TEA) were extracted by the method used for the disulfonated dyes, but the final elution was made with 3 mL of 90% methanol in 0.01% TEA/water. After evaporation the residues were taken up in 600 p L of methanol. A 20-pL aliquot from each sample was diluted to 200 p L with 35% methanol in 0.024 M NH40Ac pH 5.5 and subjected to HPLC screening. The HPLC system used was identical with the one used for the disulfonated dyes, but a concave gradient profile (no. 7 of the solvent programmer) of 35% B to of Unknown Wastewater Samples.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
85% B in 8 min was used to resolve both mono- and disulfonated dyes. The between-run variation in retention times was less than 0.6%, so retention times could be used for a preliminary identification prior to LC/MS analysis. The remainder of the extract was evaporated to dryness and taken up in 500 pL for subsequent LC/MS and LC/MS/MS analysis. For conventional column LC/MS a Waters system was used consisting of two M510 pumps, an M680 flow and gradient controller, an M440 single wavelength (254 nm) detector, and an M740 data module. Samples were injected with a Rheodyne 7125 injection valve equipped with a 20-pL loop. The Altex (Berkely CA) Ultrasphere 3-pm C184.6 mm X 75 mm column was maintained at 30 "C in a constant temperature column oven. The outlet of the UV detector was connected to the heated pneumatic nebulizer interface via a 1m long, 0.1 mm i.d. stainless steel tube. The flow rate was 2 mL/min for HPLC without MS detection and 0.5 mL/min for HPLC with MS detection. A suitable gradient made up from pure acetonitrile in pump A and 0.1 M ammonium acetate in water in pump B was used for LC/MS with the heated pneumatic nebulizer (13). Microbore liquid chromatography was performed under isocratic conditions by use of one Waters M510 pump, a Waters M680 flow controller, and a 1.8-pL flow-through cell installed in the Waters M440 UV detector. The UV detector was omitted in on-line micro LC/MS to avoid unacceptable chromatographic band broadening. Samples were injected with a Rheodyne 7520 valve using a 0.2-pL or l.0-pL sample loop. Microbore columns used were Shandon 1 mm X 100 mm Hypersil3-pm CH18and Phase Sep (Queensferry,UK) 1mm X 100 mm SpheriSorb 3-pm ODS2 Cis. A Sciex (Thornhill, Ontario, Canada) 6000E triple quadrupole mass spectrometer data system combination equipped with an atmospheric pressure ion source was used for LC/MS. Ions were sampled from the source into the vacuum system through a 100 pm i.d. orifice in the tip of a cone pointed toward the ion source region. The area in front of the orifice was flushed with highpurity dry nitrogen gas which acts as a curtain to keep solvent vapor and contaminants away from the conical orifice. Argon was used for collision induced dissociation (CID) in the second quadrupole, at 300 X 10l2atoms/cm2 target gas thickness. The collision energy was 120 eV in the laboratory frame. The Sciex heated pneumatic nebulizer (13) was operated at 265 "C indicated heater temperature, 5.0 bar nebulizer gas pressure, and 1.5 L/min make-up gas flow with 0.5 mL/min LC effluent flow rate. Under these conditions the vaporized eluent and sample mixture exits the heated quartz tube at a measured temperature of 150 "C. Nitrogen gas boiled off from liquid nitrogen was used as the nebulizing and make-up gas. A 99.999% purity nitrogen gas purchased from Airco (Murry Hill, NJ) was used as the curtain gas. The ion evaporation LC/MS interface (Sciex,Thornhill, Ontario, Canada) induced charge on HPLC effluent droplets formed by pneumatic nebulization (14). This interface handles conventional reversed-phase HPLC flow rates without any supplemental heat or restrictive orifice. This LC/MS interface was used to evaluate the analytical potential for determining the monoand disulfonated azo dyes used in this study. The HPLC conditions used were 20-40% acetonitrile/O.l M ammonium acetate in water maintained at a flow rate of 1 mL/min. The home-made "ion spray" interface is a modified combined ion evaporation/electrospray interface (15) for microbore LC/MS. The LC eluent was a mixture of acetonitrile and 0.001 M ammonium acetate in water. Separation took place under isocratic conditions at 40 pL/min flow rate. The effluent was transferred to the ion source via a 1m long, 50 pm i.d. fused silica capillary.
RESULTS AND DISCUSSION Isolation of dyes from food, gasoline, and wastewater has been done by solid phase extraction (7,10, 16) and ion pair extraction (17). Solid phase extraction was chosen for the isolation of dyes from water samples. HPLC separation of ionized solutes requires ionic additives in the eluent to prevent peak tailing and ensure reproducible retention times (18). The separation of a mixture of nine azo dyes shown in Figure 1 demonstrates that ammonium acetate is a suitable additive
2649
4
m 4
m m
a
a
u
-
N
Tine < n i n >
Flgure 1. Separation of sulfonated azo dyes by HPLC: UV detection at 254 mn; 4.6 mm X 75 mm Ultrasphere 3-Mm CI8column; flow rate 2 mL/min, gradient from 25% to 50% acetonitrile in 0.1 M ammonium
acetate in water.
to allow for rapid HPLC screening of these compounds. Under the conditions of HPLC separation given in Figure 1, filament-off thermospray LC/MS mass spectra could be observed for monosulfonated azo dyes but not for disulfonated azo dyes. After limited success from a short investigation by thermospray LC/MS either with or without filament induced ionization, we focused our attention upon atmospheric pressure ionization using three different LC/MS interfaces. These included the Sciex heated pneumatic nebulizer (13),the Sciex ion evaporation interface (14),and the home-made ion spray interface. The latter is a modified combined ion evaporation/electrospray device (15). Heated Pneumatic Nebulizer. The heated pneumatic nebulizer interface is constructed as a removable probe for the API source. The liquid HPLC effluent is fed through a pneumatic nebulizer and the aerosol travels through a heated quartz tube inside the probe (13). The vapor of sample and solvent is ionized by a high-voltage corona discharge which initiates chemical ionization between reactant ions generated from the LC eluent and sample molecules. In the presence of ammonium acetate the acetate ion a t m / z 59 is the dominant negative reactant ion, while the ammonium ion and its clusters with acetonitrile are the positive reactant ions. Negative ion mass spectrometry is preferred for sample molecules having acidic functional groups. Table I gives the negative ion APCI spectra of some monosulfonated azo dyes. These azo dyes all show the [M - HI- ion of the free acid as the base peak together with fragment ions of low relative abundance. LC/MS with an HPLC column showed that the sodium ion, which was originally associated with the acid dye, exchanges with protons of ammonium acetate. The resulting protonated acid dye can then be chromatographically resolved from the sodium-bound dimer (CH3C00CH3COONa, m / z 141) and trimer (CH3C00-.(CH3COONa)2,m / z 223). This
2650
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
Table I. Negative Ion APCI Spectra"of Monosulfonated Azo Dyesb
[M - HIm/z % 327 410
100
acid red 88
TICP
377
100
acid red 151
431
100
acid black 52 (very impure mixture)
438
100
m/z
%
238 252 207 222 349 277 215 201 217 218 240 283 328 410 213 230 254 314
30 10 5 10
100
(2 isomers)
375
LC/MS
2 5 4 nm
others
acid orange 7 acid red 337
acid yellow 151 (sulfonamide)
LC/UV
60
10
20 10 70 70 60 40 20 30 20 30 50 100 20
Mass range m / z 200-500. *Introduced via LC column; M refers to free acid or free ligand of a metal complex.
,
,
,
,
I
2
4
6 8 1 0 min
2
4
6
i 8
1
0 niin
a mixture of two isomers: sample size, 2 pg: heated pneumatic nebulizer with corona discharge atmospheric pressure negative ion chemical ionization. Figure 2. On-line LC/UV/MS of acM red 88,
221
I
Table 11. Negative Ion APCI Spectra"of Disulfonated Azo Dyesb
[M - HIm/z %
acid red 1
464
40
acid black 1
571
80
acid blue 113
636
Mass range m/z 200-700. ers to the free acid.
others m/z
%
422 446 214 224 242 258 400 422 298 311
40 100 15 20 10 25 10 100 100 10
Introduced via LC column, M ref-
sodium-proton exchange is not observed under flow injection analysis (FIA)conditions wherein the dye is injected into the eluent stream with MS detection without the benefits of an HFLC column. This appeared to be favorable for the LC/MS sensitivity for azo dyes which was better by elution through a column than by flow injection in the same eluent stream. Positive ion spectra were very weak or undetectable under the same LC eluent conditions. Disulfonated azo dyes are more difficult to analyze by mass spectrometry than the monosulfonated azo dyes. No ions were observed by either filament-off or filament-on thermospray LC/MS of these compounds in our hands. However, recent results by Vestal on an improved thermospray system have been obtained (19). The heated nebulizer negative ion APCT spectra are given in Table 11. The [M - HI- ion of the free acid is present in the spectrum of acid red 1 and acid black 1 together with abundant fragment ions. Acid blue 113 did not show a n [M - HI-ion but did show a fragment ion at mlz 298. The observed fragment ions may be due to thermal degradation in the heated nebulizer. The UV and ion current traces in Figure 2 demonstrate that the heated nebulizer interface and lip1 source do not impair the chromatographic separation in spite of the large dimensions of the source (22 cm diameter, 12 cm deep) and the low volatility of the samples. A common problem in LC/MS by any form of direct liquid introduction is the high background spectrum below m/z 150.
Figure 3. Daughter ion spectrum of the m / z 377 ion of acid red 88 (first isomer): collision gas, argon: collision energy, E,, = 120 eV.
The observation of sample fragment ions at low mass is made possible by CID in the second quadrupole of a triple quadrupole mass spectrometer. Figure 3 shows the daughter ion spectrum of mlz 377, or [M - HI- of the free acid of acid red 88, where cleavage of the azo linkage gives m / z 206 and 221 as is also observed in FAB spectra (9). The route proposed for the formation of m / z 221 is given in Figure 3. The fragment at m / z 221 is an odd-electron ion and its formation from an even-electron (M- H)- ion is made possible by the energy deposition from a collision with one or more argon atoms. The ion a t mlz 222 in Table I1 in contrast is an even-electron ion which is probably formed by the unimolecular dissociation of a metastable ion having a low internal energy. The same argument holds for the oddelectron ion at m / z 206 in the CID daughter ion spectrum as opposed to m / z 207 in the LC/MS mass spectrum of acid red 88 given in Table 11. Thermal degradation of acid red 88 into aminonaphthalenesulfonic acid and naphthalenesulfonic acid in the heated quartz tube of the pneumatic nebulizer followed by ionization may also explain m / z 201 and 222 in Table 11. Vigorous proof for either thermal degradation or unimolecular fragmentation is beyond the scope of the present investigation.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
I
OH
1
I
I
Figure 4. Parent ion spectrum of m / z 80, SO3'-, of acid red 88 (first isomer): collision gas, argon: collision energy, E,, = 120 eV.
01.
h
i
. i .k e. , i . *,e -
I
- l E
e
2
4
6
e
10
E"D4TICN TIM tHI"ES)
Figure 5. Total ion current profiles of LC/MS and LC/MS/MS of a mixture of five monosulfonated azo dyes, extracted from fortified Cayuga Lake water: sample size, 1-2 pg per component: gradient elution: flow rate, 0.5 mL/min; (1) orange 7, (2) red 337, (3, 4) red 88, (5) red 151; collision gas, argon: collision energy, E, = 120 eV; heated pneumatic nebulizer with APCI.
Most useful is the SO3'- ion a t m/z 80, a fragment ion common to sulfonated dyes. The parent ion spectrum of m/z 80 shown in Figure 4 gives the peaks observed in the single quadrupole mass spectrum of Table I. Parent ion scans on fragment ions common to a class of compounds offers a means of screening for such compounds in mixtures. A parent ion scan of m / z 80 will thus indicate the presence of sulfonated azo dyes and any other compound or ion that fragments to m / z 80 present in the mixture. Figure 5A gives the total ion current trace for the full-scan heated nebulizer LC/MS analysis of an extract of Cayuga Lake water fortified with acid orange 7, acid red 337, acid red 88, and acid red 151. The chromatographic peaks between 1and 3 min are due to unknown compounds extracted together with the azo dyes from the lake water. The total ion current trace in Figure 5B recorded by full spectrum parent ion scanning of SO3* at m / z 80 demonstrates the selectivity for sulfonated aromatics. In the case of monosulfonated azo dyes the sensitivity for clearly recognizable peaks in TIC traces is about 100 ng per component for LC/MS and 1 pg for LC/MS/MS using the heated nebulizer LC/MS interface. The sensitivity for the disulfonated azo dyes acid red 1 and acid black 1 is approximately 2 pg on column for a recognizable peak in the LC/MS TIC trace. Ion Evaporation. Pneumatic nebulization of liquids in an electric field produces charged droplets. The emission of ions from these droplets is a mild ionization process for polar and ionic samples and has been called ion evaporation (14). Since sulfonated azo dyes exist as ions in solution, ion evaporation should be well suited for mass spectrometry of these
2651
compounds. Indeed monosulfonated azo dyes gave [M-HIof the free acid and disulfonated azo dyes gave [M - 2HI2as the base peak together with a very weak [M - HI- ion. It should be noted that the disulfonated dye acid blue 113 failed to show a [M - HI- ion when introduced via the heated pneumatic nebulizer. However, with the ion evaporation LC/MS no heat is applied and thermal degradation of acid blue did not take place. Very little or no fragmentation was observed in all ion evaporation LC/MS mass spectra. Acid yellow 151, a cobalt complex of a sulfonamide could not be observed by ion evaporation LC/MS. Apparently, it is not ionized in solution, but the [M - HI- ion of the sulfonamide was easily detected in the above-described APCI process. The ion currents recorded from ion evaporation were 10- to 100-fold lower than those obtained by using the heated pneumatic nebulizer which precludes the application of ion evaporation to real-life samples. Ion Spray. An LC/MS interface has been constructed (15) that combines some characteristics of electrospray (20). These features include low flow rates, a spray capillary at high voltage with respect to the ion source walls, with some details of the ion evaporation interface, and pneumatic nebulization in an electric field. Since it is neither pure electrospray nor identical with ion evaporation we have chosen the short name "ion spray". Its operating principle is emission of ions from charged droplets. As expected, the mass spectra by ion spray of sulfonated azo dyes are identical with those obtained by the ion evaporation LC/MS interface. Sensitivity for sulfonated azo dyes by ion spray, however, is appreciably higher than by ion evaporation or by APCI using the heated pneumatic nebulizer. Approximately 10 ng of a monosulfonated azo dye is sufficient for a full-scan mass spectrum and clearly recongizable peaks in total ion current traces. Liquid-phase ionization by ion spray or ion evaporation shows less than a factor of 10 difference in sensitivity between all mono- and disulfonated azo dyes together. Gas phase ionization was 20-50 times less sensitive for disulfonated azo dyes compared with monosulfonated azo dyes. An important observation was that sensitivity for sulfonated azo dyes by liquid phase ionization is strongly reduced when ammonium acetate at a concentration of 0.01 M or more is added to the LC eluent to improve LC peak shapes. The same effect was reported for thermospray LC/MS of ionic samples (15). A 0.001 M ammonium acetate ion concentration gave adequate chromatographic separation without sacrificing sensitivity. Ion spray and ion evaporation have a common mild ionization mechanism that generates few or no fragment ions. Again, tandem mass spectrometry can be used for structure elucidation and selective identification in these instances. The SO3'- ion discussed above is a characteristic fragment in the MS/MS determination of sulfonated azo dyes. Selected reaction monitoring (SRM) of the fragmentation reactions [M - HISO3'- of each sulfonated azo dye allows selective detection of each dye. Figure 6 shows the SRM profiles for monosulfonated azo dyes extracted from fortified Cayuga Lake water. Although the selectivity was increased, the absolute sensitivity dropped by transmission and scattering losses in two quadrupole mass filters and the collision region. The solid phase extraction procedure described above and used for fortified Cayuga Lake water was applied to fortified municipal wastewater. Each wastewater sample contained one dye from the series given above. After solid phase extraction the retention times by HPLC suggested the presence of acid black 1,acid orange 7, acid red 337, acid red 151, and possibly acid red 88. Acid red 151 was easily distinguished by its long retention time. Acid red 88 was ruled out by the
-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
2652
all four extracts under the same conditions. Gradient elution is preferred. Figure 7 demonstrates the capability of ion spray LC/MS for identifying azo dyes in real-world samples.
IONS/SEC 758811
i l 327 410 377 431
m/z
x:., 417
3. 4
,
,
.
,
,,
~,
,, ,
---
CONCLUSION m/z
. )
80 80 80 80
Registry No. Acid red 337,12270-02-9;acid red 151,6406-56-0; acid black 52, 5610-64-0;acid red 88,1658-56-6;acid yellow 151, 12715-61-6;acid orange 7, 633-96-5; acid red 1, 3734-67-6; acid black 1, 1064-48-8;acid blue 113, 3351-05-1.
,
II 4 I i:, I
.
2
4
6
8 TIME
RETENTION
( m i d
Flgure 6, Ion spray LC/MS/MS of a mixture of five monosulfonated azo dyes (see Figure 5) extracted from fortified Cayuga Lake water, selected reaGtlon monituing of [M - HISO;-: cowiskn gas,argon; collision energy, E,, = 120 eV; sample size, approximately 20 ng of each component; liquid phase ionization by ion spray; 1 mm i.d. X 100 mm HypersU 3-Mm C18column; flow rate, 40 pL/mln; 30% acetonltrlle, 7 0 % 0.001 M ammonium acetate in water.
-
2oo'ooolG 100.000
i
40'0001 n
20.000
i
Atmospheric pressure ionization mass spectrometry allows for the LC/MS determination of nonvolatile azo dyes. The ionization methods described herein together with collision induced dissociation constitute a flexible, sensitive, and specific identification procedure.
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LITERATURE CITED (1) McGovern, P. E.; Michei, R. H. Anal. Chem. 1985, 5 7 , 1514A. (2) Cook, A. M.; Thurnheer, T.; Kdrler-Staub, D.; Galii, R.; Grossenbacher, H.; Swiss Biotech 1988, 4 , 15-17. (3) Rinde, E.; Troll, W. J . /Vat/. Cancer fnst. 1975, 55. 181-182. (4) Ventkatakaman. K. The Analytical Chemistry of Synthetic Dyes ; Wk ley-Interscience: New York, 1977. (5) Bailey, J. E. Anal. Chem. 1985, 5 7 , 189-196. (6) Tincher, W. C.; Robertson, J. R. 1982. 74, 269-275. (7) Youngless, T. L.; Swansinger, J. T.; Danner, D. A.; Greco, M. Anal. Chem. 1985, 57. 1894-1902. (8) Mathias, A.; Williams, A. E.; Games, D. E.; Jackson, A. H. Org. Mass Spectrom. 1978. 7 7 . 266-270. (9) Monaghan. J. J.; Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. M. Org. Mass Spectrom. 1983, 78, 75-82. (10) Voyksner, R. D. Anal. Cbem. 1985, 57, 2600-2605. (11) Betowski. L. D.; Ballard, J. M. anal. Chem. 1984, 5 6 , 2604-2607. (12) Voyksner, R. D. Monthly Technical Progress Report for EPA Contract No. 68-03-3122, Research Triangle Park, NC, Nov 1984. (13) Covey, T. R.; Lee, E. D.; Bruins, A. P.; Henion, J. D. Anal. Chem. 1988, 58. 145lA-1461A. (14) Iribarne, J. W.; Dziedzic. P. J.; Thomson, B. A. Int. J . Mass Spectrom. Ion Phys. 1983, 5 0 , 331-347. (15) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem., preceding paper In this issue. (18) McKone, H. T.; Ivie, K. J . Chsm. Educ. 1980, 5 7 , 321-322. Blnkle, B.; Scheller, A. Fresenius' Z . Anal. Chem. 1986, (17) Lehman, 0.; 323, 355-358. (18) Rudzlnski, W. E.; Bennett, D.; Garcia, V. J . Llq. Chromafogr. 1982, 5 , 1295-1312. (19) Vestal, M. L. 4th LC/MS Workshop, Montreux, Oct 22-24, 1986. (20) Whkehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 5 7 , 675-679. (21) Schmelzeisendedeker, G.;Roilgen, F. W.; Wirtz, H.; Vogtle, F. Org. Mass Spectrom. 1985, 2 0 , 752-756. (22) Llnn, C. K.; Peters, T. J. J . Chromatogr. 1984, 376,397-406.
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Figure 7. Ion spray LC/MS of two monosutfonated azo dyes extracted from fortifled wastewater, selected ion monitorlng of [M - HI- ions of orange 7 and red 337: from top to bottom, standard injection (10 ng) extract of regular wastewater, and extract of forwled wastewater; #quid phase ionization by ion spray; 1 mm X 100 mm Spherisorb 3-pm column; flow rate, 40 pL/min; 25% acetonitrile, 7 5 % 0.001 M ammonlum acetate In water. absence of its two isomers and by selected ion monitoring. Figure 7 shows the SIM traces for a standard solution and two of the extracts. Isocratic conditions in our micro LC system were not suitable for separation and presentation of
RECEIVED for review December 3, 1986. Accepted July 24, 1987. A.P.B., L.O.G.W., and J.D.H. thank the Environmental Monitoring & Support Laboratory of the Environmental Protection Agency, Cincinnati, OH, for financial support (Grant No. CR-811661-10-0).
