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Introduction The following describes the analysis of explosive compounds in soil and water samples using LC/MS/MS. Instrumentation consisted of a High Performance Liquid Chromatography (HPLC) system equipped with an autosampler and coupled with a triple quadrupole mass spectrometer using negative ion Atmospheric Pressure Chemical Ionization (APCI). The mass spectrometer was tuned and optimized to use two Multiple Reaction Monitoring (MRM) transitions per analyte. The most sensitive MRM transition was used for quantitation while the second one was used for detection confirmation. The Lower Limit of Quantitation (LOQ) for each analyte was set based on a measured signal to noise of 10:1. After sample preparation was performed all analytes could be detected at levels ranging from 0.05 µg/L to 2.0 µg/L in water samples and 25.0 µg/kg to 1000 µg/kg in soil samples. Detailed detection limits for each analyte are given in Table 4. Additionally, studies were performed to show that ion suppression is not present for these samples when using APCI. Overview The analysis of explosive residues in ground water and soil samples is an ever increasing challenge. Due to the production and expulsion of military weaponry, there is an increasing need for accurate and definitive detection of explosive compounds and their degradation products. Both military installations and local municipalities surrounding these installations are concerned about these hazardous compounds entering water supplies. 1, 2, 3 The standard technique used for the analysis of these compounds has been HPLC with UV detection, following the guidelines set by USEPA method 8330. 4 Although this method has been successful, so far, there are several drawbacks. The lack of sensitivity of UV detection requires a costly and time consuming concentration step during preparation of samples. Trace Level Analysis of Explosives in Ground Water and Soil API 3200™ LC/MS/MS system APPLICATION NOTE Explosive Analysis

Trace Level Analysis of Explosives in Ground Water and Soil · of explosive compounds in soil and ... Explosives compounds, by design, are unstable, thermally labile molecules. Because

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Page 1: Trace Level Analysis of Explosives in Ground Water and Soil · of explosive compounds in soil and ... Explosives compounds, by design, are unstable, thermally labile molecules. Because

Introduction The following describes the analysis of explosive compounds in soil and water samples using LC/MS/MS. Instrumentation consisted of a High Performance Liquid Chromatography (HPLC) system equipped with an autosampler and coupled with a triple quadrupole mass spectrometer using negative ion Atmospheric Pressure Chemical Ionization (APCI). The mass spectrometer was tuned and optimized to use two Multiple Reaction Monitoring (MRM) transitions per analyte. The most sensitive MRM transition was used for quantitation while the second one was used for detection confirmation.

The Lower Limit of Quantitation (LOQ) for each analyte was set based on a measured signal to noise of 10:1. After sample preparation was performed all analytes could be detected at levels ranging from 0.05 µg/L to 2.0 µg/L in water samples and 25.0 µg/kg to 1000 µg/kg in soil samples. Detailed detection limits for each analyte are given in Table 4.

Additionally, studies were performed to show that ion suppression is not present for these samples when using APCI.

Overview The analysis of explosive residues in ground water and soil samples is an ever increasing challenge. Due to the production and expulsion of military weaponry, there is an increasing need for accurate and definitive detection of explosive compounds and their degradation products. Both military installations and local municipalities surrounding these installations are concerned about these hazardous compounds entering water supplies. 1, 2, 3 The standard technique used for the analysis of these compounds has been HPLC with UV detection, following the guidelines set by USEPA method 8330.4 Although this method has been successful, so far, there are several drawbacks. The lack of sensitivity of UV detection requires a costly and time consuming concentration step during preparation of samples.

Trace Level Analysis of Explosives in Ground Water and Soil

API 3200™ LC/MS/MS system

APPLICATION NOTE Explosive Analysis

Page 2: Trace Level Analysis of Explosives in Ground Water and Soil · of explosive compounds in soil and ... Explosives compounds, by design, are unstable, thermally labile molecules. Because

Experimental Conditions Water and soil samples were collected randomly around the Denver and San Francisco Bay area.

Water Samples Water samples were prepared by Solid Phase Extraction (SPE). The SPE cartridge pretreatment consisted in washing with 10 mL of methanol followed by 50 mL of water. One Liter of sample was loaded onto the Waters Porapak RDX phase cartridge at a rate of 3.0 liters an hour. The SPE cartridge was then dried by pulling nitrogen through it. Then, 5.0 mL of acetonitrile was loaded onto the phase and allowed to sit for 5 minutes before eluting off at gravity flow. When 5.0 mL of sample were obtained, 5.0 mL of 1.0% acetic acid in water was finally added. Please note that a low concentration of acetic acid was used in the final extract to preserve the presence of Tetryl. At high pH Tetryl quickly degrades and is inconsistently detected.

More importantly, UV detection is not selective and requires a second analysis using a separate HPLC column. Even after this second analysis, there is still possibility of false positive detection. This is no longer acceptable, especially with the increasing terror threat, and forensic and criminal laboratories need to quickly and unambiguously screen for common explosive compounds at trace levels.

Presented is a sensitive method for the detection of a wide range of nitroaromatic and nitroamine compounds by LC/MS/MS using an API 3200™ system with negative ion Atmospheric Pressure Chemical Ionization (APCI). By using MS/MS detection the concern of false positive detections is virtually eliminated.

Figure 1. Trace level multi-component analysis of nitroaromatic and nitroamine compounds.

TAbLE 1: MONITOrEd MrM TrANsITIONs. *rATIO = QuANTIFILEr MrM ArEA/QuANTIFILEr MrM ArEA.

Analyte retention Molecular Observed Quantifier Qualifier Expected Time (min) Weight Precursor Ion MrM MrM MrM ratio*

HMX 3.67 296 M+CH3COO- 355/46 355/147 1.76

RDX 5.51 222 M+CH3COO- 281/46 281/59 2.77

1,3-Dinitrobenzene 8.96 168 M- 168/46 168/138 1.74

1,3,5-Trinitrobenzene 7.38 213 M- 213/183 213/63 3.95

2,4,6-Trinitrotoluene 11.15 227 M- and (M-H)- 226/46 227/210 2.81

Tetryl 9.13 287 M-NO2 241/213 241/196 1.2

4-Amino-2,6-Dinitrotoluene 11.68 197 (M-H)- 196/46 196/136 16.1

2,-Amino-4,6-Dinitrotoluene 12.43 197 M- and (M-H)- 196/136 197/46 4.28

2,6-Dinitrotoluene 12.95 182 M- 182/46 182/152 1.87

2,4-Dinitrotoluene 13.48 182 M- and (M-H)- 182/46 181/46 0.93

PETN 17.19 316 M+CH3COO- 375/62 375/46 1.90

2,2,4,4,6,6- Hexanitrodiphenylamine 13.97 439 M- 438/226 438/362 1.42

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Soil Samples Soil samples were prepared by weighing out 2.0+/-0.1g of dry soil into a 40.0 mL amber glass vial. Then 5.0 mL of acetonitrile were then added to the samples. After sonication at 6.0°C +/- 2.0°C for 18 hours, the samples were centrifuged for 3 minutes. Finally, 3.0 mL of the supernatant were removed and combined with 3.0 mL of 1% acetic acid.

Calibration Curve A calibration curve was prepared in 50:50 water:acetonitile over a range of 0.49µg/L – 1.0 mg/L. Serial dilutions were performed starting with a 1.0 µg/mL standard.

All standards of explosive analytes (Accustandard Inc.) were prepared in 100% acetonitrile and kept a 4°C.

Final extracts were analyzed on an API 3200™ LC/MS/MS system using Atmosphere Pressure Chemical Ionization (APCI) in negative ion mode. Evaluation was performed using Multiple Reaction Monitoring (MRM). The two most sensitive MRM transitions were monitored for each analyte.

INsTruMENT PArAMETErs:

Curtain Gas (N2): 10

CAD gas (N2): 6

Gas 1: 35

Interface Heater: On

Desolvation Temperature: 350°C

Nebulizer Current: -8

Dwell time for each MRM transition: 50 ms

TAbLE 2

Time (Min) A (%) b (%)

0 42 58

15 42 58

18 0 100

20 0 100

20.1 42 58

25 42 58

A Shimadzu SCL10Avp Integrated HPLC equipped with two LC10ADvp pumps was used for chromatographic analysis. Eluent A consisted of H20 with 0.5mM Ammonium and eluent B was Methanol. 20 µL were injected onto a Phenomex Ultracarb ODS 20, 250x4.6 mm, 5 µm particle size column. (Phenomenex, Torrance, CA, USA) The sample was eluted using the gradient described in Table 2 with a 1.0 mL/min flow rate.

results and discussion Explosives compounds, by design, are unstable, thermally labile molecules. Because of this they can be difficult to analyze in a laboratory setting. In addition, this class of compounds requires a special set of condition for sensitive and stable ionization.

As shown in table 1, the most intense precursor ion for each compound varied in nature. As expected, some of them produced a [M-H]- quasi molecular ion, while some produced acetate adducts, i.e. [M+CH3COO]- , or an intense M-• molecular anion. The latter species can be produced in negative ion APCI by electron capture or by charge exchange with ionized ambient gas and/or solvent.5, 6

In the case of Tetryl, the [M-NO2]- fragment was chosen as this species is more easily produced.

Because of possible thermal degradation of compounds with APCI, chromatographic separation of most analytes is important. For example, TNT can thermally degrade into 2,6 and 2,4-Dinitrotoluene. Therefore, if these compounds were to co-elute, TNT would show up in the MRM transition of the Dinitrotoluene compounds as a potential false positive.

By chromatographically separating the compounds and monitoring two MRM transitions the possibility of false positives is eliminated.

The most sensitive MRM transition was used for quantitation (quantifier) while the second one was used for confirmation (qualifier). Ratios of the measured area counts of both MRM transitions were then monitored. Table 1 shows the transitions used for each analyte along with the observed ion ratio. The proper ion ratio for each explosive analyte was determined by averaging the MRM ratio for each analyte in each point of the calibration curve. Calibration curve information for all transitions is shown in Table 3.

All analytes have a calculated correlation coefficient of 0.99 or greater for both quantifier and qualifier MRM transition. A linear regression fit was used for all analytes with a weighting factor of 1/x. If a weighting factor was not used the curve was then forced through the origin. Curve type was based upon best fit of the calibration data.

Water and soil samples were then extracted according to the procedure presented. For each sample a matrix spike was prepared to ensure extraction efficiency. All samples were analyzed following the calibration curve. A Continuing Calibration Verification (CCV) sample was analyzed after the samples to verify that the instrument had maintained calibration. Figure 1 shows the results of a matrix sample spiked with a 500 µg/L standard mixture.

The Lower Limit of Detection (LOD) for each analyte was determined with a signal to noise ratio of 3:1 in both the quantitation ion and the confirmation ion. The Lower Limit of Quantitation (LOQ) was determined with a signal to noise ratio of 10:1. See Table 4 for detailed detection limits in both soil and water matrixes. Ion ratios were compared to those obtained from the calibration curve for confirmation and good correlation between standards and spiked matrices was found.

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From all of the samples collected, no explosive compounds were detected above the established LOD. Matrix spike tests on all of the samples showed recoveries of 80 – 120% indicating good recoveries from the sample preparation procedure. Figure 2 shows a water sample spike with all target explosives.

Ion Suppression Studies Although it is less common to see ion suppression in APCI negative ion mode, it’s important to test it especially if inconsistent or unreliable results are noticed.

To monitor possible ion suppressions, a post column infusion of a 1.0 µg/mL standard solution of all compounds of interest was performed during the injection of 20 µL of matrix (water or soil) on the column. The infusion of the standard solution creates a constant signal on the related MRM traces. If the sample matrix were to cause ion suppression a dip in the signal should be recorded.

The test was performed on three different matrices: one water matrix (North Table Mountain Irrigation Canal, Golden, CO, see figure 3) and two soil matrices (North Table Mountain East Entrance Location, Golden, CO and San Francisco Bay Coyote Park, CA; see figure 4 and 5 respectively) In all cases no significant ion suppression was observed.

TAbLE 3: CALIbrATION CurvE INFOrMATION

Analyte Name Correlation regression Weighting Coefficient Fit

HMX 0.9994 Linear 1/x

HMX CONFIRM 0.9998 Linear 1/x

RDX 0.9995 Linear 1/x

RDX CONFIRM 0.9993 Linear 1/x

1,3-Dinitrobenzene 0.9996 Linear 1/x

1,3-Dinitrobenzene

CONFIRM 0.9996 Linear 1/x

1,3,5-Trinitrobenzene 0.9986 Linear 1/x

1,3,5-Trinitrobenzene CONFIRM 0.9978 Linear 1/x

2,4,6-Trinitrotoluene 0.9994 Linear 1/x

2,4,6-Trinitrotoluene CONFIRM 0.9988 Linear 1/x

Tetryl 0.9919 Linear Thru Zero None

Tetryl CONFIRM 0.9923 Linear Thru Zero None

4-Amino-2,6-Dinitrotoluene 0.9999 Linear 1/x

4-Amino-2,6-Dinitrotoluene

CONFIRM 0.9992 Linear 1/x

2-Amino-4,6-Dinitrotoluene 0.9997 Linear 1/x

2-Amino-4,6-Dinitrotoluene CONFIRM 0.9997 Linear 1/x

2,6-Dinitrotoluene 0.9992 Linear 1/x

2,6-Dinitrotoluene CONFIRM 0.9989 Linear 1/x

2,4-Dinitrotoluene 0.9952 Linear 1/x

2,4-Dinitrotoluene CONFIRM 0.9984 Linear 1/x

PETN 0.9961 Linear 1/x

PETN CONFIRM 0.9945 Linear 1/x

Hexyl 0.9978 Linear 1/x

Hexyl CONFIRM 0.9998 Linear 1/x

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Conclusion An LC/MS/MS method for the analysis of trace level explosive compounds has been presented. This method has proven to be a sensitive and specific method that is an improvement from the traditional LC/UV technique. With the added specificity of Multiple Reaction Monitoring and the ion ratio confirmation using two transitions the concerns of false positive detection has virtually been eliminated. It has also been shown that ion suppression in water and soil matrixes is not a concern with APCI ionization for the tested compounds.

Samples that were collected for this study did not show any residual explosives. Matrix spike test on the matrixes indicated that all analytes can be recovered with good sensitivity and show an accurate ion ratio for confirmation.

Future work is planned on an API 5000™ LC/MS/MS system with Atmospheric Pressure Photo Ionization (APPI) to develop an ultra sensitive direct injection water analysis method. This method would eliminate any sample preparation procedure and allow for higher throughput.

Authors C. Borton and L. Olson Applied Biosystems/MDS Sciex, Foster City, California

Acknowledgements The authors would like to thank Dr. Paul Winkler of GEL Analytical in Golden, Colorado for help with sample preparation during this study. We would also like to thank our colleagues at Applied Biosystems/MDS Sciex for their thoughts and contributions.

Figure 2. Water sample spike at 0.05 to 0.10 ug/L depending on analyte.

TAbLE 4: WATEr sAMPLE sPIkE AT 0.05 TO 0.10 µg/L dEPENdINg ON ANALyTE

Analyte Water LOd Water LOQ soil LOd soil LOQ (g/L) (µg/L) (µg/kg) (µg/kg)

HMX 0.01 0.04 5.0 17.5

RDX 0.01 0.04 5.0 17.5

1,3-Dinitrobenzene 0.02 0.07 10.0 35.0

1,3,5-Trinitrobenzene 0.01 0.04 5.0 17.5

2,4,6-Trinitrotoluene 0.01 0.04 5.0 17.5

Tetryl 0.04 0.14 20.0 70.0

4-Amino-2,6-Dinitrotoluene 0.02 0.07 10.0 35.0

2,-Amino-4,6-Dinitrotoluene 0.01 0.04 5.0 17.5

2,6-Dinitrotoluene 0.08 0.30 40.0 140

2,4-Dinitrotoluene 0.08 0.30 40.0 140

PETN 0.4 1.40 200.0 700

2,2,4,4,6,6- Hexanitrodiphenylamine 0.02 0.07 10.0 35.0

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references 1 “Innovative Uses of Composting: Composting of Soils Contaminated by Explosives”, http:/www.epa.gov/ epaoswer/non-hw/compost/explos.pdf

2 “Hazard Assessment of Munitions and Explosives of Concern (MEC) Workgroup”, http://ww.epa.gov. swerffrr/documents/hazard_assess_ wrkgrp.htm

3 6.3: Explosives, Provides information about explosive, http://www.epa.gov/ ttn/chief/ap42/ch06/final/c06s03.pdf

4 Explosives Residue Standard Operating Procedure, http://www.epa. gov/Region2/desa/hsw/sop8330.pdf

5 Harrison, Alex. G, “Chemical Ionization Mass Spectrometry” 1983

6 Tiina J Kauppila, Tapio Kotiaho, Risto Kostiainen and Andries P Bruins, “Negative ion-atmospheric pressure photoionization-mass spectrometry”, Journal of the American Society for Mass Spectrometry, Volume 15, Issue 2, February 2004, Pages 203-211

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Figure 3. Water sample: North Table Mountain Irrigation Canal, Golden, CO – No significant ion suppression observed from sample matrix.

Figure 5. Soil sample: San Francisco Bay Coyote Park, CA – No significant ion suppression observed from sample matrix.

Figure 4. Soil sample: North Table Mountain East Entrance Location, Golden, CO – No significant ion suppression observed from sample matrix.