Anal. Chem. 1999, 71, 4142-4147
Multicomponent Quantification of Diastereomeric Hexosamine Monosaccharides Using Ion Trap Tandem Mass Spectrometry Heather Desaire and Julie A. Leary*
College of Chemistry, University of California, Berkeley, California 94720-1460
A rapid means of stereochemical differentiation and quantification for the hexosamine monosaccharides was achieved using electrospray ionization quadrupole ion trap mass spectrometry. The hexosamine monosaccharides, glucosamine, galactosamine, and mannosamine, were derivatized with [Co(DAP)2Cl2]Cl, and the complex [Co(DAP)2(HexNH2)]Cl was generated. Subjecting this complex to collision-induced dissociation provided a unique product ion spectrum for each of the diastereomeric monosaccharide complexes, thus differentiating the stereoisomers. Furthermore, the stereoisomers were quantified. This was achieved by using the relative abundances of product ions from pure standards and using these values to determine the ratio of isomeric products in a mixture. The utility of this quantification method was demonstrated by successfully determining the composition of two- and three-component mixtures of the hexosamines. Mass spectrometry is becoming an increasingly powerful tool in the biological sciences, particularly in determining structural features of complex biopolymers, including proteins, DNA, and carbohydrates. Yet, differentiation of isomeric structures is often difficult or impossible when using mass spectrometry. In carbohydrate analysis, this issue is particularly significant, as many possible isomeric structures can exist for any oligosaccharide. Conditions that differentiate some isomeric features of oligosaccharidessincluding sequence of monosaccharide units, linkage position, and anomeric configurationscan be achieved by a variety of mass spectrometric methods.1-13 Recent studies have shown * Corresponding author: (phone) (510) 643-6499; (fax) (510) 642-9295; (e-mail)
[email protected]. (1) Egge, H.; Peter-Katalinic, J. Mass Spectrom. Rev. 1988, 6, 331. (2) Zhou, Z.; Ogden, S.; Leary, J. A. J. Org. Chem. 1990, 55, 5444. (3) Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 5964. (4) Staempfli, A.; Zhou, Z.; Leary, J. A. J. Org. Chem. 1992, 57, 3590. (5) Garozzo, D.; Impallomeni, G.; Montaudo, G.; Spina, E. Rapid Commun. Mass Spectrom. 1992, 6, 550. (6) Garozzo, D.; Giuffrida, M.; Impallomeni, G.; Ballistreri, A.; Montaudo, G. Anal. Chem. 1993, 62, 279. (7) Fura, A.; Leary, J. A. Anal. Chem. 1993, 65, 2805. (8) Hayes, G. R.; Williams, A.; Costello, C. E.; Enns, C. A.; et al. Glycobiology 1995, 5, 227. (9) Reinhold: V.; Reinhold: B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772. (10) Cancilla, M. T.; Penn, S. G.; Carroll, J. A.; Lebrilla, C. B. J. Am. Chem. Soc. 1996, 118, 6736. (11) Sible, E. M.; Brimmer, S. P.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1997, 8, 32.
4142 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
that the stereochemistry of hexose and N-acetylhexosamine monosaccharides has been differentiated, when the monosaccharides are derivatized with metal-ligand complexes prior to the MS/MS experiments.14,15 As a continuation of this work, we have developed a method that will also differentiate the amino sugars, glucosamine (GlcNH2), galactosamine (GalNH2), and mannosamine (ManNH2), which appear in many biologically significant glycosaminoglycans, such as heparin.16 Furthermore, these monosaccharides are present in virtually every oligosaccharide sample that is subjected to total acid hydrolysis, because the ubiquitous N-acetylhexosamines are reduced to hexosamines during hydrolysis conditions.17 Total acid hydrolysis is a common first step in oligosaccharide structural determination, and once hydrolyzed, the monomeric components must be separated prior to analysis. If MS could be used to quantify these isomeric mixtures, the separation step could be avoided. Using multicomponent quantification we have discovered a novel way to successfully quantify these isomers without separation. A few research groups have shown that mixtures of the structural isomeric compounds leucine and isoleucine may be analyzed using blackbody infrared radiative dissociation (BIRD) or mass-analyzed ion kinetic energy spectroscopy/collisioninduced dissociation (MIKES/CID) experiments.18,19 Yet, to date quantification of biologically relevant stereoisomers has not been achieved. Furthermore, it is not obvious how the BIRD or MIKES/ CID methodologies could be extended to allow for the analysis of more than two isomeric components at once. In research presented herein, a method for multicomponent quantification is utilized.20 The method, which was originally used on electron impact mass spectra of oil distillates, is applied to ESI MS/MS spectra of three biologically relevant monosaccharide diastereomers. The method is rapid and accurate, and most importantly, it is applicable to any series of isomeric compounds. Furthermore, while a mixture of three diastereomers is used here, multicom(12) Gaucher, S. P.; Leary, J. A. J. Am. Soc. Mass Spectrom 1999, 10, 269. (13) Fangmark, I.; Jansson, A.; Nilsson, B. Anal. Chem. 1999, 71, 1105. (14) Gaucher, S. P.; Leary, J. A. Anal. Chem. 1998, 70, 3009. (15) Desaire, H.; Leary, J. A. Anal. Chem. 1999, 71, 1997. (16) Chaplin, M. F., Kennedy, J. F., Eds. Carbohydrate Analysis; Oxford: New York, 1994. (17) Fukuda, M., Kobata, A., Eds. Glydobiology; Oxford: New York, 1993. (18) Schnier, P. D.; Williams, E. R. Anal. Chem. 1998, 70, 3033. (19) Krishna, P., Prabhakar, S. Vairamani, M. Rapid Commun. Mass Spectrom. 1998, 12, 1429. (20) Roboz, J. Introduction to Mass Spectrometry: Instrumentation and Techniques; Interscience: New York, 1968. 10.1021/ac990553w CCC: $18.00
© 1999 American Chemical Society Published on Web 08/25/1999
Scheme 1
ponent quantification can be applied to isomeric mixtures that contain more than three isomers as well. EXPERIMENTAL SECTION The complex [CoCl2(DAP)2]Cl, where DAP is diaminopropane, was prepared by standard methods.21 Synthesis of the Hexosamine Complexes. A methanolic solution of [CoCl2(DAP)2]Cl and N(CH2CH3)3 (0.02 M in each reagent) was prepared, and a separate, aqueous solution of the monosaccharide (HCl salt) (0.023 M) was prepared. Each solution was made daily. The methanolic solution, 5 µL (100 nmol) and 4.35 µL of the aqueous solution (100 nmol) were combined and vortexed for 20 s. The solution was then microwaved for 90 s at 90% power in a capillary tube. The mixture was diluted to a final concentration of 50 pmol/µL and injected via direct infusion into the mass spectrometer at a flow rate of 5 µL/min. Mass Spectrometry. The mass spectrometer used was a Finnigan LCQ, which employs a quadrupole ion trap and an electrospray ionization source. No modifications to the instrument were made. For all experiments, the capillary was heated to 150 °C, and the spray voltage was maintained at 5.2 kV. It was empirically determined that placing the ESI probe as close to the spray shield as possibleswithout arcingswas desirable. The Finnigan LCQ has three possible settings for the ESI probe, which determine the distance of the ESI source to the heated capillary. The middle of the three settings provided the most reproducible results in our laboratory, when a 5.2-kV spray voltage was employed. (On this setting, the source protruded approximately 1.6 cm from the ESI flange.) Optimization of the ion of interest was initially completed using the automatic tuning parameter on the instrument. This tune file was then loaded for every experiment. To avoid space charge effects, the number of ions in the trap was regulated by the automatic gain control, which was set to 5 × 107 counts for MS1 scans and 4 × 107 for MS/MS experiments. The injection time, however, was not allowed to (21) Bailar, J. C.; Work, J. B. J. Am. Chem. Soc. 1946, 68, 232.
Figure 1. Isomeric monosaccharides.
exceed 400 ms for any scan. Typical ion injection times for MS/ MS experiments were 200-400 ms. The isolation window for the ion of interest was 3.0 mass units for all MS/MS experiments. The excitation voltage was 0.37 V (peak to peak), applied for 1000 ms. The q value was maintained at 0.25, and the mass range was m/z 150-400. Under these conditions, a good signal-to-noise ratio was established. A typical signal for the MS/MS experiments was 1 × 105 counts, with electron multiplier gain set to -750 V. Each spectrum consisted of 40 scans, and each scan comprised 3 “microscans” for a total of 120 scans averaged for each measurement. RESULTS AND DISCUSSION Differentiation of Diastereomers. Structural differences among the three biologically relevant hexosamines, GlcNH2, GalNH2, and ManNH2, occur at the C2 and C4 positions (Figure 1). Each monomer is reacted with 1 equiv of [CoIII(DAP)2Cl2]Cl to produce an ion at m/z 384 as a major product of the reaction, shown in Scheme 1 and Figure 2. The ion at m/z 384 represents the complex CoIII(DAP)2(hexosamine - 2H)+ shown in Scheme 1 and depicts the GlcNH2 diastereomer, which we propose to be an octahedral structure. While this complex has not been fully characterized, the proposed structure is consistent with Bunel’s work, which shows that complexes of the type CoIII(en)2(hexosamine) are octahedral, with the saccharide coordinated to Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
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Figure 2. MS of a typical reaction mixture. Ion of interest, m/z 384, is [Co(DAP)2(GalNH2)]+.
Figure 3. MS/MS of m/z 384. (A) ManNH2; (B) GlcNH2; (C) GalNH2.
the metal at C1 and C2.22,23 Furthermore, the structure is consistent with the MSn data presented here and all previously isolated compounds from our laboratory.14,15,24-26 The MS/MS spectra resulting from CID of the m/z 384 precursor ion for each of the three diastereomers are shown in Figure 3. The major product ion in each spectrum is observed at m/z 310 and corresponds to the loss of a neutral DAP ligand. In (22) Bunel, S.; Ibarra, C.; Moraga, E.; Andrei, B.; Bunton, C. A. Carbohydr. Res. 1993, 244, 1. (23) Bunel, S.; Ibarra, C.; Moraga, E.; Calvo, V.; et al. Carbohydr. Res. 1993, 239, 185. (24) Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1996, 118, 3293. (25) Smith, G.; Pedersen, S. F.; Leary, J. A. J. Org. Chem. 1997, 62, 2152. (26) Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1998, 120, 13046.
4144 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
addition to this common product ion, differentiating ions specific to each diastereomer are also observed and are listed with the relative abundances in Table 1. As seen in Figure 3A, CID of the ManNH2 complex produces only one additional product ion, m/z 295, with any appreciable abundance. Given this odd mass loss of 89 Da and the lack of any high-resolution mass measurements to date, a likely composition for this loss is C3H7NO2. A possible explanation for this loss involves a simultaneous loss of C2H5NO (2-amino-1-ethenol) and CH2O (formaldehyde). A similar dissociation was observed previously when complexes of the type Co(DAP)2(HexNAc)+ underwent CID.15 However, without extensive labeling experiments or high-resolution measurements of the hexosamine complex, we cannot unambiguously assign this
Table 1. Comparison of Dissociations for the Ion m/z 384
glucosamine
m/z 366
m/z 326
m/z 295
- H2O (3.3%)
- C2H2O2 (24.8%) or - C2H4NO
- C3H7NO (4.0%)
galactosamine - H2O (21.9%) mannosamine
Table 2. Reproducibility of Relative Abundances m/z 295
326
366
ManNH2
40.4 40.7 40.7 39.2 39.6 38.9 37.9 38.8
2.01 2.22 2.17 2.29 2.18 2.47 2.11 2.44
0.35 0.32 0.35 0.27 0.26 0.31 0.32 0.38
av SD
39.53 1.01
2.24 0.16
0.32 0.04
- C3H7NO (39.5%)
product ion loss. Nonetheless, the ion at m/z 295 is a predominant ion in the ManNH2 spectrum but is obviously absent in the spectrum generated from the GalNH2 complex. In addition to the ions at m/z 310 and 295, two additional ions are detected in the MS/MS spectrum of the GlcNH2 diastereomer. The ion at m/z 366, although of relatively low abundance, corresponds to loss of H2O, while a unique ion at m/z 326 is also found in Figure 3B. This 58 Da loss could represent two possible compositional losses: acetylene diol (C2H2O2) or a radical loss of C2H4NO. On the basis of the structure of a hexosamine, the loss of acetylene diol is highly unlikely. The loss of C2H4NO seems unusual given the fact that a radical loss has been observed only once previously from a metal-ligand-saccharide complex.25 In this complex, however, a cobalt(III) metal is stabilized by two sites of deprotonation, affording a species with an overall charge of +1 (Scheme 1). The radical loss may be a favorable dissociation process because it could cause reduction at the metal center to a +2 oxidation state, leaving only one site of deprotonation and minimizing overall charge separation. Labeling experiments and Fourier transform ion cyclotron resonance (FT-ICR) studies are currently planned in order to better understand the processes associated with these losses, which are stereoselective in the gas phase. Figure 3C, representing the GalNH2 complex, is the simplest of the three spectra in that only the losses of H2O and DAP are observed. Given the data generated and presented in Table 1, it is evident that the MS2 spectra may be used to distinguish each of the three diastereomers provided that a relative abundance of >3% is used as the criterion to establish the presence or absence of an ion, as was established in our previous work.27 It is also important to note that the relative abundances measured in each MS/MS spectrum of these complexes are very reproducible from run to run and day to day as described and discussed below. Multicomponent Quantification. By carefully adjusting several experimental parameters, deviation in the product ion intensities could be minimized significantly. The important factors that contribute to the accuracy and reproducibility of the experiments were as follows: solution preparation, activation parameters (voltage, activation time, q value, scan range), and spray voltage. Furthermore, results were most reproducible when samples were analyzed immediately after derivatization. The instrumental and experimental conditions were carefully optimized in order to maximize reproducibility of product ion abundances, and these conditions are described in detail in the Experimental Section. Using the conditions described above, each monosaccharide was derivatized and analyzed a total of eight times over the period of 1 week, and the results are shown in Table 2. For the pure (27) Koenig, S.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1998, 9, 1125.
GlcNH2
4.05 3.96 3.99 4.03 3.98 4.03 3.65 4.03
25.0 24.6 24.4 25.3 24.4 24.9 24.6 25.3
3.41 3.3 3.34 3.42 3.01 3.37 3.38 3.42
av SD
3.96 0.13
24.8 0.37
3.33 0.14
GalNH2
0
av SD
0
22.4 22.2 22.5 21.3 21.3 21.6 21.6 21.9 21.9 0.49
monosaccharides, variation in intensity of each product ion is quite small, as evidenced by the low standard deviations for the product ions. Similar experiments conducted in our laboratory suggest that the intensities of the product ions do not drift, even over periods of time as long as 6 weeks, when samples are run on the same instrument under identical conditions. Generally, when samples are quantitatively analyzed by mass spectrometry, a calibration plot is generated and quality control standards and unknown samples are then tested using the calibration curve. For example, Krishna et al. plotted the intensity of product ions versus the mole percent of derivatized leucine and isoleucine, which they then quantified.19 A similar method could be used here. Calibration curves for GlcNH2/ManNH2 mixtures, GlcNH2/GalNH2 mixtures, and ManNH2/GalNH2 mixtures could be generated in a fashion similar to Krishna et al., and samples with any two of the three biologically relevant hexosamines could be quantified. Unfortunately, producing these calibration plots is somewhat labor intensive. Furthermore, it is not obvious how this method could be applicable to analyzing a mixture with all three diastereomers present.By using a novel method of quantitative analysis, the composition of two- or threecomponent mixtures of hexosamines could be determined without calibration plots. In this method, the intensities of the three product ions of interest, (m/z 295, 326, and 366) for the pure monosaccharides (Table 2) can be used to determine the composition of a mixture of monosaccharides. To demonstrate how the intensities of the pure product ions can be used to determine the composition of a mixture, the three-component mixture of Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
4145
Table 3. Three-Component Mixtures of Hexosamines rel abund from spectra
% computed
% normalized
% error
mixture
m/z 295
m/z 326
m/z 366
gal
man
glc
gal
man
glc
% actual gal/man/glc
gal
man
glc
1 1 2 2 3 3 4 4
13.8 14.5 10.7 11.0 8.3 8.4 32.2 32.6
9.2 9.0 13.5 13.7 5.8 5.9 4.3 4.7
9.1 8.7 8.0 7.7 13.6 14.7 2.8 3.2
35.8 34.1 28.2 26.6 58.6 63.6 10.2 11.8
31.5 33.3 21.8 22.5 18.7 19.0 80.5 81.4
34.3 33.1 52.5 53.2 21.7 22.0 10.0 11.4
35.2 33.9 27.5 26.0 59.2 60.8 10.1 11.3
31.0 33.1 21.3 22.0 18.9 18.2 79.9 77.8
33.8 32.9 51.2 52.0 21.9 21.0 9.9 10.9
33/33/33 33/33/33 25/25/50 25/25/50 60/20/20 60/20/20 10/80/10 10/80/10
2.2 0.9 2.5 1.0 0.8 0.8 0.1 1.3
2.0 0.1 3.7 3.0 1.1 1.8 0.1 2.2
0.8 0.1 1.2 2.0 1.9 1.0 0.1 0.9
GlcNH2/GalNH2/ManNH2 at a 1:1:1 concentration ratio will be used as an illustrative case. For this mixture, we predicted that the relative abundance of the ion m/z 295 is simply the average of the relative abundances of the product ion m/z 295 for each of the pure monosaccharides. That is,
0.33 × 295glc* + 0.33 × 295gal* + 0.33 × 295man* )
In the example above, each diastereomer represented one-third of the total monosaccharide concentration; therefore, the coefficient in front of each term on the left side of the equations is 0.33. By adjusting this coefficient, the system of three equations can be applied to any mixture of monosaccharides. For example, the system of equations below could be used to describe a mixture that is 50% GlcNH2, 25% GalNH2, and 25% ManNH2:
295(1:1:1)** mixtures of pure components
or
0.33 × 4.0* + 0.33 × 0* + 0.33 × 39.5* ) 14.5%** where * indicates relative abundances for the product ion m/z 295, as listed in Table 2, and ** indicates predicted relative abundance for the product ion m/z 295 for the 1:1:1 mixture. Thus, 14.5 is the predicted percent relative abundance for m/z 295 in the three-component mixture. The experimentally observed values obtained from the MS2 spectra were 13.8 and 14.5% (Table 3, lines 1 and 2), which agree quite well with the theoretical relative abundances. The intensities of the other two relevant product ions, m/z 326 and 366, could also be predicted in a similar fashion. The relative abundances for m/z 326 as obtained from Table 2 are 24.8, 2.3, and 0% for GlcNH2, ManNH2, and GalNH2, respectively. The average of these three values, 9.0%, agrees well with the observed values, 9.2 and 9.0%, for the relative abundance m/z 326 in the 1:1:1 mixture GlcNH2/GalNH2/ManNH2 (Table 3). Likewise, using the same method, the predicted value for the relative abundance of m/z 366 for the 1:1:1 mixture is 8.5% and the experimentally obtained values were 9.1 and 8.7%. Thus, the following three equations can be used to describe the expected relative abundances of the ions m/z 295, 326, and 366.
mixtures of pure components 0.33 × 295glc* + 0.33 × 295gal* + 0.33 × 295man* ) 0.33 × 326glc* + 0.33 × 326gal* + 0.33 × 326man* ) 0.33 × 366glc* + 0.33 × 366gal* + 0.33 × 366man* )
theor abund (%)
exptl measd (%)
(m/z 295) 14.5
0.5 × 295glc* + 0.25 × 295gal* + 0.25 × 295man* ) 0.5 × 326glc* + 0.25 × 326gal* + 0.25 × 326man* ) 0.5 × 366glc* + 0.25 × 366gal* + 0.25 × 366man* )
theor abund (%)
exptl measd (%)
(m/z 295) 11.9
10.7, 11.0**
(m/z 326) 13.0
13.5, 13.7**
(m/z 366) 7.2
8.0, 7.7**
where * indicates relative abundances for the product ions m/z 295, 326, and 366 as listed in Table 2 and ** indicates relative abundances for the 50:25:25 mixture. (See Table 3, lines 3 and 4.) Again, the agreement of the expected values with the experimentally obtained values suggests that this method may be a reasonable way to predict product ion abundances for these mixtures. Not only can this system of equations be used to predict the intensities of product ions for a three-component mixture of monosaccharides but it can also be used to determine the percentage of each monosaccharide in an unknown mixture, once the intensities for each of the product ions are measured. For example, the product ions m/z 295, 326, and 366 for mixture 3 (Table 3) had experimentally measured relative abundances of 8.3, 5.8, and 13.6%, respectively. Thus, the following three equations could be solved for a, b, and c, (where a is % GlcNH2, b is % GalNH2, and c is % ManNH2, in the mixture).
Measured abundances a × 295glc + b × 295gal + c × 295man ) 8.3%
(1)
13.8, 14.5
a × 326glc + b × 326gal + c × 326man ) 5.8%
(2)
(m/z 326) 9.0
9.2, 9.0
a × 366glc + b × 366gal + c × 366man ) 13.6%
(3)
(m/z 366) 8.5
9.1, 8.7
where * indicates relative abundances for the product ions m/z 295, 326, and 366 as listed in Table 2. 4146 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
Solving the equations for a, b, and c, using Mathematica Version 3 for Unix, gives the percent of each monosaccharide in the mixture. For the three-component mixture described above (mixture 3) the values of a, b, and c, were 58.6, 18.7, and 21.7%,
Table 4. Two-Component Mixtures of Hexosamines rel abund from spectra
% computed
% normalized
% error
mixture
m/z 295
m/z 326
m/z 366
gal
man
glc
gal
man
glc
% actual gal/man/glc
gal
man
glc
1 2 3 4 5 6 7 8 9
12.0 21.0 28.7 4.0 1.2 1.0 26.2 23.7 4.1
20.2 14.0 8.8 18.2 13.8 5.9 1.4 1.2 0.2
2.9 2.0 1.2 7.6 12.9 16.8 6.3 10.5 19.8
0.6 0.4 0.2 23.5 50.6 73.4 27.7 47.3 90.2
22.4 47.9 69.7 1.3 -2.7 0.1 66.4 60.0 10.4
79.6 52.2 29.0 73.3 55.7 23.7 -0.2 -0.7 0.0
0.6 0.4 0.2 24.0 47.6 75.5 29.4 44.1 89.7
21.8 47.7 70.5 1.3 0.0 0.1 70.6 55.9 10.3
77.6 52.0 29.3 74.4 52.4 24.4 0.0 0.0 0.0
0/20/80 0/50/50 0/67/33 25/0/75 50/0/50 75/0/25 33/67/0 50/50/0 90/10/0
0.6 0.4 0.2 1.0 2.4 0.5 3.9 5.9 0.3
1.8 2.3 3.5 1.3 0.0 0.1 3.9 5.9 0.3
2.4 2.0 3.7 0.3 2.4 0.6 0.0 0.0 0.0
respectively. (Table 3, mixture 3, “% computed”). At this point, no restrictions have been placed on the coefficients a, b, and c. That is, the solutions to the equations may be negative, and a + b + c will likely not equal 100%. To improve the accuracy of the results, the values obtained from eqs, 1-3 are normalized to meet the following two criteria: First, a + b + c ) 100%, and second, no value may be less than zero. By normalizing the computed values for mixture 3 described above, the percent of each monosaccharide in the mixture is adjusted to 59.2, 18.9, and 21.9% for GalNH2, ManNH2, and GlcNH2, respectively, as indicated in Table 3 under “% normalized”, mixture 3. Finally, one can observe that these values agree quite well with the actual percentages of each component (Table 3, mixture 3, “% actual”) and that the error for each monosaccharide is quite small: 0.8, 1.1, and 1.9% for GalNH2, ManNH2, and GlcNH2, respectively (Table 3, “% error”). Four different mixtures were analyzed using this method (Table 3), and each mixture was analyzed twice. Error values for each measurement are quite small, generally less than 4% for each component. In Table 3, each mixture contained all of the three possible components; yet this technique does not require all three components to be present. Table 4 shows many examples of twocomponent mixtures that have been quantified using the method above. For the two-component mixtures, as with the threecomponent mixtures, the same equations (1-3) as described earlier were used. For example, mixture 1 (Table 4) initially contained 80% GlcNH2 and 20% ManNH2. When this mixture was derivatized with [Co(DAP)2Cl2]Cl and MS/MS was performed on the ion m/z 384, the three product ions of interest, m/z 295, 326, and 366, had the relative abundances 12.0, 20.2, and 2.9%. By substituting these values into eqs 1-3, the unnormalized values for GalNH2, ManNH2, and GlcNH2 were determined to be 0.6, 22.4, and 79.6%, respectively (Table 4, mixture 1, “% computed”). After normalization of these values, the experimentally determined
ratios for each monosaccharide were 0.6% to 21.8% to 77.6% (Table 4, mixture 1, “% normalized”), which is in close agreement with the actual values of 0, 20, and 80% for GalNH2, ManNH2, and GlcNH2, respectively. (Note: negative values for monosaccharide concentration were normalized to zero.) Other examples of twocomponent mixtures are also listed in Table 4. Again, for the twocomponent mixtures, error was consistently small. CONCLUSION Mixtures of hexosamines can be quantified using the multicomponent quantification method described. This method is unique in that it does not require the use of calibration plots. It is the first method to quantify diastereomeric mixtures, and it is the first mass spectrometric quantification of a three-component, isomeric mixture using electrospray ionization. Furthermore, this study introduces a derivatizing reagent, [Co(DAP)2Cl2]Cl, which can be used to produce metal-hexosamine complexes that provide unique product ion spectra, differentiating the diastereomeric hexosamines. This method of hexosamine differentiation and quantification is quite advantageous because the derivatization is rapid and inexpensive, and orthogonal separation techniques are not required. Isotopic labeling experiments to determine the origins of the discriminating product ions are underway, and application of the methodology on oligosaccharide samples isolated from biological systems will be presented separately. ACKNOWLEDGMENT The authors gratefully acknowledge NIH grant GM47356 for financial support. H.D. also thanks Dr. Glenn Smith for helpful discussions and suggestions. Received for review May 25, 1999. Accepted July 19, 1999. AC990553W
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