2702
Anal. Chem. IQSS, 58,2782-2784
Determination of Nitrogen- 15 Enrichment of Nitrate and Nitrite Using Thermospray Liquid Chromatography-Mass Spectrometry L. R. Hogge,* R. K. Hynes, and L. M. Nelson
Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan S7N OW9, Canada
M. L. Vestal Chemistry Department, University of Houston, Houston, Texas 77004
A new method has been developed for the determlnatlon of percent 15N enrlchment In NO2- and NO3- that Is both rapld and sendtlve. NO2- and NO3- separation was done by use of reverse-phase llquld chromatography wHh a moblle phase conslstlng of ammonium acetate wlth tetrabutylammonlum acetate as the Ion palr reagent. The % "N In lsotoplcally enrlched anlons was determined by udng thennospray llquld chromatography-mass spectrometry (LC-MS). Enrlchment values were obtalned on as little as 20 ng of N02--N/20 gL and 4 ng of N03--N/20 pL. The linear regresslon curve was plotted for the observed enrichment vs. the actual enrichment over the range from the natural abundance to 25% enrlchment for each anion and the equaHon for the Hnear regredon -I-0.7577 for NO2- and y = 0 . 9 5 1 ~ calculated ( y = 1 . 1 6 ~ 0.447 fOr NO,). The COdtldW~tOf detemdnatkn for the N02and NO3- curves was 0.988 and 0.987, respectlvely. The dynamlc range of the method was found to be &table for the range of NO2- and NO3- expected In plant xylem sap. Xylem sap samples obtalned from NO3- fed pea, P / m sat/vum cv. Homesteader (3.9 pg of N03--N/mL, 24.7% 15N), contalned a 15N enrlchment of 25.4% f 0.7%.
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The determination of nitrate (NO,-) and nitrite (NO,-) in soils and plant tissues is very important in the study of nitrogen metabolism in plants and microorganisms (I). The use of 15N-enriched forms of N to trace the movement of these anions from soil into the plant provides more precise data on uptake and partitioning within the plant as well as on possible pathways of metabolism for the added N. Several techniques for the measurement of NO3- and NOC are available and have been described in detail (2,3). These methods include colorimetric assays, ion-specific electrodes, gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), and conventional mass spectrometric methods. Colorimetric assays and ion-specific electrodes are useful; however they do not provide isotope ratio information. GC and GC-MS methods require conversion of NO3- and NOz- to compounds that can be separated and detected by the gas chromatograph ( 4 , 5 ) . Unfortunately, conventional mass spectrometric methods require a substantial amount of N (about 1 mg) in the material to be examined and the analyses are time-consuming since the 16N forms must first be converted to ammonium (NH4+)and then oxidized to Nz (6). Clearly, none of the currently available methods offer a rapid screening technique that provides isotopic ratio information. Determination of NO3- and NOz- by high-performance liquid chromatography (LC) has been available for several years; this method also does not allow isotope ratio deter-
minations (7).However, the combination of liquid chromatography and mass spectrometry (LC-MS), although not previously shown to be useful for inorganic anions, has provided rapid analyses including isotopic ratio information for many organic compounds (8). The recent development of thermospray LC-MS (9,IO) prompted the authors to investigate this new technique as a more sensitive and rapid method for the measurement of Nos- and NOz- as part of an ongoing study of nitrogen metabolism in a plant-soil column system. EXPERIMENTAL SECTION Materials and Instrumentation. Standard samples of NO2and NO3- were prepared by dissolving known amounts of 99 atom % 15N sodium nitrite and potassium nitrate (MSD Isotopes, Montreal, Canada) in deionized water. All enrichment values expressed in the text are atom % excess. A set of standards was prepared for each anion. Each standard set consisted of five samples containing 0, 5, 10, 15, and 25% lSN enrichment, respectively, at a concentration of 1.3 ng of NO,--N/pL and 0.9 ng of NO,--N/pL. LC analyses were done with a Waters system consisting of two pumps (Models 590 and 510) interfaced to a Model 680 gradient controller and Model 441 absorbance detector operating at 214 nm. The samples were injected on a 10-cm Whatman Partisil 5 ODS-3 C18 reverse-phase column in 20-wL aliquots with a Rheodyne Model 7010 injector. Isocratic analyses were performed using a mobile phase consisting of 0.01 M ammonium acetate and 0.005 M tetrabutylammonium acetate, at a flow rate of 1.5 mL/min, adjusted to pH 6.0. For LC-MS analyses, the ammonium acetate concentration was increased to 0.1 M (vide infra). LC-MS analyses were done with a Model 3300 Finnigan GCMS system retrofitted with a Vestec thermospray interface (Veatec Corp., Houston, TX). Data were acquired with a Model 2300 Finnigan Incos Data System. Negative ion mass spectra were obtained in the multiple ion detection mode (MID) by scanning mass intervals of m/z 40-45 and mlz 60-65 repeatedly every 0.6 S.
Plant Growth. Pea seeds (Pisurn satiuum, cv. Homesteader) were planted in laboratory soil columns located in a controlled environment chamber (Enconaire, Winnipeg, Canada) with a 16-h photoperiod and day-night temperatures of 20 and 15 "C, respectively. A mixture of fluorescent and incandescent lights supplied photon flux densities (400-700 nm) of 400 pmol quanta/(m2/s). The columns were watered continuously at a flow rate of 7.5 mL/h with a nutrient solution supplemented with KN03, 3.9 pg of N03--N/mL (11). When the plants were 4 weeks old, the columns were watered as above, but with 25% 15N-enriched KNOB,3.9 pg of NO;-N/mL replacing the unenriched KNOB. At 5 weeks the plants were decapitated and latex tubing was slipped over the stem. Sap that accumulated in the latex tubing was transferred to 1-mL Reacti-vials and frozen until the analysis of NO< and NO3- was performed. RESULTS AND DISCUSSION To facilitate thermospray LC-MS analyses, the LC conditions used in Eek and Ferrer's earlier work were modified
0003-2700/86/0358-2782$01,50/0Published 1986 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
NO;
NO;
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TIME (minutes)
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Flgure 1. Chromatogram showing the LC separation and detection of NO2- and NO3- (UV absorbance detector at 214 nm) using the modified mobile phase.
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ACTUAL % ENRICHMENT
Flgure 3. Plot of observed MS for NO2- (a) and NO3- (b).
~ 5 6
3808
wo 332 1844 3156 SCAN TIME (minutes)
4208
Figure 2. Thermospray LC-MS total ion chromatogram showing the separation of NO,- and NO,-.
(7). Ammonium acetate buffer and tetrabutylammonium acetate ion pair reagent were used in place of ammonium phosphate and tetrabutylammonium phosphate, respectively, as the former compounds are sufficiently volatile to minimize contamination of the ion source. After several weeks of use the ion source showed no lack of performance due to contamination from the mobile phase. This modified mobile phase did not noticeably affect the chromatographic separation or the sensitivity of the analyses for NO, and NO; as is shown in Figure 1. Analyses of these anions were done using a concentration of 0.01 M ammonium acetate for UV detection and 0.1 M for thermospray LC-MS detection. The higher concentration of ammonium acetate was necessary for thermospray analyses as the ion current in the thermospray-mass spectrometer interface is proportional to the square root of the concentration of ions in solution (12). However, this concentration (0.1 M) of buffer was not suitable for UV detection as it gave a high background UV absorbance a t 214 nm. Chromatographic separation of the anions was not affected noticeably by the change in concentration of ammonium acetate. Figure 2 shows the thermospray LC-MS total ion chromatogram for 25% enriched NO2- and NO3- (25 ng of N02--N and 17 ng of N03--N). The observed enrichment (mean of four samples) was 25.4% for NO3- and 25.3% for NO2- compared to actual enrichment values of 24.9% and 25.4%, respectively. It should be noted that although the percent enrichment values showed good agreement for both anions, the ion current for NOz- is about 20% that of NO3-. Dilution studies of this mixture showed that isotopic enrich-
YO enrichment vs. actual % enrichment
ment determinations were possible for 20 ng of N02--N and 4 ng of N03--N. Comparison of Figure 1,the chromatogram obtained from UV detection, with Figure 2 indicates that the difference in sensitivity for NOz- and NO; exists in the thermospray ionization process. Direct injection (without the LC column) of equal amounts of NOz- and NO3- gave the same difference in sensitivities. The reason for the discrepancy in sensitivity between NOz- and NO3- is not known a t this time. The reliability of the method for the range of enrichments from natural abundance to 25% enrichment was tested. This was done by performing thermospray LC/MS analysis on standards of NOz- and NO3- with known amounts of 15N enrichment and plotting these observed enrichment values vs. the actual enrichment. Five replicate thermospray analyses were carried out on each sample and the % I5N enrichment was calculated from the mass spectral data. Figure 3 shows the plots of the observed enrichment vs. actual enrichment for NO2- and NO3-, respectively. For the NO2- enrichment standard curve, the equation for the linear regression line was y = 1.165% 0.7577. The coefficient of determination value, R2, was 0.988. For the NO3- standard curve the equation was y = 0 . 9 5 1 ~ 0.447 and R2 was 0.987. To determine if the dynamic range of this technique is sufficient for the concentration of the anions expected in plant sap, analyses of 25% 15N enriched NOz- and NO3- standard solutions ranging in concentration from 25 ng to 1 pg and 17 ng to 0.7 pg NO,-N and N03--N, respectively, were performed. The expected ratio of percent enrichment of each anion was observed at all of the levels tested (Table I). The value of LC in this type of analysis is shown in Figure 4, the thermospray total ion chromatogram of a plant nutrient solution. This figure shows the single ion traces for the ‘*NOT, 15NO;, l4NO3-,and l5No3- ions which are m/z 46,47, 62, and 63, respectively. A third compound with common ions of m j z 46 and 63 is shown eluting just ahead of NO, and NO3-. The value of the LC to this method is clearly demonstrated in the
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2784
ANALYTICAL
CHEMISTRY,
VOL. 58, NO. 13, NOVEMBER 1986
Table I. Analysis of Different Concentrations of Enriched NOz- and NO,- by Thermospray LC-MS" anion
ng of N02--N o r N03--N injected obsd enrichment
NOz'
25 101 203 1015 17 69 139 693
Nos-
24.6 24.2 23.0 22.7 25.0 26.8 26.8 25.3
i 0.6 f 0.1 i 0.6 f 0.4 f 0.2 zk 0.5 f 0.4 i 0.4
" A c t u a l % I5N enrichment of NOz- a n d NO3- was 24.89 a n d 25.41, respectively. Values are t h e mean of four samples f standa r d error. 0
/l
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2
4
6
8
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TIME (minutes) Figure 5. LC chromatogram of plant xylem sap showing the presence
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46'
of NO3-.
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K 50
100
150
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250
S C A N NUMBER
Flgure 4. Single lon traces for '%OF, I 5 N O ~'%O
