TECHNICAL NOTE 10654

Negative Ion Chemical Ionization (NCI) studies using the Thermo Scientific ISQ 7000 single quadrupole GC-MS system

Author Goal Amit Gujar To demonstrate NCI capabilities of the Thermo ScientificTM ISQTM 7000 single Thermo Fisher Scientific, quadrupole GC-MS system with methane reagent gas, attainable typical Austin, TX, USA instrument detection limits (IDLs), and the effect of the ion source temperature and reagent gas flow rates on NCI mass spectra.

Keywords Introduction Negative ion chemical Negative ion chemical ionization (NICI or NCI) is a popular ionization ionization (NICI or NCI), IDL, technique for gas chromatography-mass spectrometry (GC-MS) that is Octafluoronaphthalene, ISQ frequently used for analyzing electrophilic molecules of environmental 7000, gas chromatography-mass concern such as brominated flame retardants (BFR)1 and chlorinated spectrometry pesticides.2 It is also a popular technique in steroid and drug analysis after derivatization of the analyte with compounds that add electronegative atoms to the molecule.3 The advantages the NCI technique offers are high sensitivity and selectivity for electrophilic molecules in complex matrices.

Electron capture (as opposed to true negative ionization involving high energy electron-molecule reaction) is the main mechanism in formation of negative ions in the mass spectrometry technique as shown below.4

AB + e- (~0.1 eV) ABˉ˙ (Resonance electron capture)

AB + e- (0-15 eV) � A˙ + Bˉ (Dissociative electron capture)

AB + e- (>10 eV) �Aˉ + B+ + e- (Ion-pair formation)

� This is why NCI is also referred to as electron capture This technical note provides details on the methane-NCI negative ionization (ECNI). ECNI results from resonance capability of the ISQ 7000 single quadrupole GC- capture of low-energy (thermalized) secondary electrons MS system. It shows the IDL for the system using formed as a result of electron ionization (EI) of a reagent octafluoronapthalene (OFN) and the effect of ion source gas such as methane contained in a closed ion source. temperature and methane gas flow rates on the mass Other examples of commonly used reagent gases include spectral pattern and chromatographic peak shape for isobutane and ammonia. 4TMS-derivative of benzo[a]pyrene-7,8,9,10-tetrol (BaPT) and PFPA-HFIP-derivatized 11-nor-9-carboxy-THC. NCI is considered to be a soft ionization technique BaPT is an important metabolite in toxicokinetic studies yielding a mass spectral pattern with less fragmentation of benzo[a]pyrene, a potent carcinogenic polycyclic and where the molecular or pseudo-molecular ions are aromatic hydrocarbon (PAH).5 BaPT is routinely monitored easily identifiable. A comparison of mass spectra for in blood and urine, and due to being nonvolatile, it is EI and NCI of octafluoronaphthalene (OFN) is shown in often derivatized in order to make it appropriate for Figure 1. NCI shows significantly less fragmentation than GC analysis. 11-nor-9-carboxy-THC is an important EI with a dominant molecular ion peak (Mˉ˙) of 272. metabolite of tetrahydrocannabinol (THC) which is formed in the body and is regarded as an indicator 120 6 % of cannabis consumption. It is routinely monitored in

272 100 various biological matrices like plasma, urine and hair.

80 Hair matrix is especially challenging because of the low concentration of the metabolite in hair and small amounts 60 241 available. However, GC-MS/MS in NCI mode has been (a) 40 7 222 demonstrated to be suitable. 203 20 141 273

0 Experimental

m/z Sample and sample preparation: 1 fg/µL OFN in -20 50 75 100 125 150 175 200 225 250 275 300 isooctane (Thermo Scientific, catalog #1R6310-0101K) 120 % was used, as is, in IDL studies. Other concentrations 272 100 shown in these studies were prepared by diluting the 80 1 fg/µL OFN solution by isooctane.

60 (b) Benzo[a]pyrenetetrol (BaPT, catalog # B205850) was 40 purchased from Toronto Research Chemicals, Inc. 20 273 (Canada). As received, BaPT (0.125 mg) was derivatized 0 by the addition of 1 mL Thermo Scientific™ MSTFA + 1%

m/z -20 TMCS Silylation Reagent (catalog # TS-48915) and heating 50 75 100 125 150 175 200 225 250 275 300 at 60 °C for 40 hours in a sealed vial. After derivatization Figure 1. (a) EI mass spectrum of Octafluoronaphthalene (b) NCI the sample was directly injected into the mass spectrum of Octafluoronaphthalene GC-MS system without further dilution. Figure 2 shows the molecular structure of BaPT and its TMS-derivatized form.

2 11-nor-9-Carboxy-Δ9-THC (catalog # T-010) was OH purchased from Cerilliant corporation (Round Rock, HO Texas) and derivatized with perfluoropropionic anyhydride (PFPA) and hexafluoroisopropanol (HFIP) using a procedure similar to that in Reference 7. The

HO chemical reaction is shown in Figure 3. Notice that the derivatized product is highly fluorinated and hence OH is ideal for analysis by GC-MS in NCI mode. Figure 4 (a) shows comparison of the EI and NCI mass spectra for the compound. Even though the EI spectrum gives a distinguishable molecular ion (m/z 640) the compound CH3 fragments into many low mass ions while the NCI H3C Si CH spectrum shows prominent M-HF (m/z 620) ions with H C 3 3 noticeably less fragmentation. CH3 O Si

H3C O

O H C 3 Si CH3 O CH3 CH3 Si H3C

H3C (b)

Figure 2. Molecular structures of (a) Benzo[a]pyrene-7,8,9,10-tetrol (BaPT) and (b) 4TMS-BaPT

Esterification OH F F F F F F F F F F F O OH F F O O F F F F Hexafluoroisopropanol (HFIP) OH O O ΔT → H3C + -H2O H C F 3 -C F COOH O H3C O F 2 5 Acylation H3C F F

F F F O CH3 CH3 F O

O 9 11-nor-9-carboxy-THC F F 11-nor-9-Carboxy-∆ -THC-PFPA-HFIP C27H27F11O5 Perfluoropropionic anhydride (PFPA) Mol.Wt. = 640.168 Da

Figure 3. Chemical reaction for derivatization of 11-nor-9-Carboxy-Δ9-THC leading to highly fluorinated analyte suitable for GC-MS NCI analysis

3 120 %

69 100

80

477 60 119 71 640 40 (a) 55 489

91 445 473 625 20 115 165 77 95 339 53 117 141 377 597 82 169 209 269 293 353 446 490 569 68 187 237 325 401 598 626 0

m/z -20 50 100 150 200 250 300 350 400 450 500 550 600 650 700 120 %

620 100 492

80

472 60

(b) 40 147 493 621

20 383 473 452 432 384 494 600 622 640 0

m/z -20 50 100 150 200 250 300 350 400 450 500 550 600 650 700

Figure 4. Comparison of the EI (a) and NCI (b) mass spectrum of PFPA-HFIP-11-nor-THC-COOH at ion source temperature of 200 °C

GC-MS conditions GC-MS system and the Thermo Scientific™ TSQ™ 9000 The ISQ 7000 mass spectrometer coupled with a Thermo triple quadrupole GC-MS/MS system does not require a Scientific™ TRACE™ 1310 Gas Chromatograph and a separate electron collector and has an integrated electron Thermo Scientific™ AI/AS 1310 Series Autosampler was lens that draws the electrons out and into the ion volume. used for the experiments. Typical autosampler, GC, and High electron energies are not optimal for this design. MS method parameters used for the OFN detection limit studies are shown in Table 1. The Thermo Scientific™ The IDL was calculated by statistical analysis using the one- ExtractaBrite™ source with CI ion volume was used. tailed Student’s t-distribution at a 99% confidence interval One important point to note is that electron energy of for eight sequential injections. The equation used is: 70 eV is used. It has been traditionally regarded that high electron energy (~500 eV) is necessary to get good IDL = t Amount %RSD, chemical ionization.8,9 This was because older filament ∗ ∗ designs used an electron trap collector where higher where: electron energies were necessary for the electrons to t = one-tailed Student’s t-value travel through the reagent gas to get to the collector. Amount = amount of analyte (injected on-column) The modern hairpin filament design of the ISQ 7000 RSD = relative standard deviation for chromatographic peak areas

4 Using one-sided Student’s t-distribution for 99% confidence Software interval for eight injections (n, degrees of freedom = 7) we Thermo Scientific™ Chromeleon™ Chromatography have t = 2.998. Thus the IDL equation becomes Data System (CDS) software, version 7.2 was used for data acquisition and analysis. The Cobra peak detection IDL = 2.998 Amount %RSD algorithm was used and the peak asymmetry (also known as tailing factor) calculation mentioned in the results ∗ ∗ The IDL for OFN was demonstrated in a SIM (selected ion below are based on the USP formula considering the monitoring) mode, monitoring m/z 272. peak width measurements at 5% of the height.

Table 1. Autosampler, GC, and MS parameters used for the OFN detection limit experiments.

AI/AS 1310 Series Autosampler Syringe 10 µL, 25 gauge, 50 mm length, cone tip (P/N 36500525) Injection volume 1 µL Plunger strokes 3 Draw speed Slow Sampling depth Bottom Pre-/post-injection dwell time 0.0 s Pre-injection solvent and cycles 0 Sample rinses 1 Post-injection solvent and cycles 0

TRACE 1310 Gas Chromatograph Column TG-SQC 15m × 0.25mm × 0.25µm (P/N 26070-1300) Liner LinerGOLD liner, splitless single taper with quartz wool, 4mm × 6.5mm × 76.5mm (453A1925-UI) SSL mode Splitless Inlet temperature 220 °C Splitless time 0.5 min Split flow 50 mL/min Septum purge flow Constant flow of 5.0 mL/min Carrier flow Constant He flow of 1.2 mL/min Oven program 45 °C (0.5 min), 40 °C/min to 190 °C (0 min)

ISQ 7000 GC-MS system Method type Acquisition – General MS transfer line temperature 250 °C Ion source temperature 200 °C Ionization mode Negative CI, 70 eV CI gas Methane CI gas flow rate 1.25 mL/min Emission current 50 µA Scan start 2.4 min Scan SIM m/z 272 Dwell time 0.1 s Tuning used Autotune_NCI

5 Results and Discussion 1 fg/µL OFN demonstrating the stability of the system. Detection limit study for OFN The %RSD of the area counts was 4%. Figure 7 shows Figure 5 shows the time and signal offset overlaid comparison of the chromatogram at various low levels chromatograms of eight 1 µL sequential injections of of OFN ranging from 1 fg to 0.05 fg on-column. Also 1 fg/µL OFN for m/z 272. The calculated IDL was 0.1 fg. shown is the blank iso-octane chromatogram. Notice the For comparison, the average calculated OFN IDL in EI peak-to-peak signal to noise ratio on the peak label. We mode using the Thermo Scientific™ Advanced Electron observe that one can easily detect OFN in NCI mode at Ionization (AEI) source was 0.64 fg.10 Figure 6 shows the levels as low as 100 ag on-column with signal to noise area count trend for over 100 sequential injections of ratio of >3, thus confirming the calculated IDL. This equates to detecting approximately 200,000 molecules.

1.0e4 2

counts 6 . 2 :

9.0e3 T R - N F

8.0e3 O

7.0e3

6.0e3

5.0e3

4.0e3

3.0e3

2.0e3

1.0e3

min 0.0e0 2.401 2.500 2.600 2.700 2.800 2.900 3.000

Figure 5. Chromatogram showing eight sequential 1 µL injections of 1 fg/µL OFN for SIM of m/z 272

OFN MS_Quantitation counts*sec

3s 4000

Mean 3500

3s

3000

2500 Area (MS Quantitation,OFN) 2000

1500

1000 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N 1fg OF N Injections

Figure 6. Area counts trend for over 100 sequential injections of 1 fg/µL OFN for SIM of m/z 272 with mean and +/- 3 standard deviations

6 8.0e3 5.0e3 counts RT=2.61; Area=3594; S/N=47.9 counts RT=2.61; Area=1907; S/N=24.1 N N

4.0e3 O F O F

6.0e3 g g 5 f . 1 f 0 3.0e3 4.0e3

2.0e3

2.0e3

1.0e3 min min 0.0e0 5.0e2 2.240 2.500 2.750 3.040 2.240 2.500 2.750 3.040

1.9e3 1.4e3 counts counts 1.8e3 RT=2.61; Area=388; S/N=5.1 1.3e3 N a n k O F

1.2e3 b l

1.6e3 g 8 1 f . i C 0 1.1e3 1.4e3

1.0e3 1.2e3

9.0e2 1.0e3 8.0e2 min min 8.0e2 7.5e2 2.240 2.500 2.750 3.040 2.240 2.500 2.750 3.040

Figure 7. Comparison of various low level OFN injections showing the ability of the ISQ 7000 to easily detect levels as low as 100 ag on-column. Also shown in the isooctane blank chromatogram.

Effect of ion source temperature and reagent of the peak as shown in Figure 10 while the effect of methane flow rates ion source temperature on peak shape for derivatized The mass spectrum in NCI mode is sensitive to mass 11-nor-9-Carboxy-Δ9-THC (Figure 11) is minimal — spectrometer run conditions such as ion source even when at much lower source temperatures. This temperature and reagent gas flow rates. Figure 8 shows shows that peak shapes are dependent on the analyte the effect of ion source temperature on the mass properties and that silylated molecules are more prone spectrum of 4TMS-BaPT at 1.5 mL/min methane flow. to adsorption effects within the ion source leading to Decreasing the ion source temperature results in lower increased tailing effects at low temperatures versus abundance of low mass ions such as m/z 284 versus non-silylated molecules (fluorinated in this instance). higher mass ones such as m/z 446. A similar trend Very low ion source temperatures (< 150 °C) are not can be seen for derivatized 11-nor-9-Carboxy-Δ9-THC recommended with methane-CI as it decreases the in Figure 9 where relative abundance of m/z 620 (M- robustness of the system due to matrix adsorption and HF) versus m/z 492 increases with lower ion source fouling of the ion volume by methane reagent ions. It is temperatures. Generally, high mass ions are preferred therefore important to find a balance amongst the ion since they give higher selectivity to compounds of source temperature, peak tailing factors and the desired interest which in turn improves the detection limits robustness for the desired application. for such compounds due to higher signal to noise ratios. An adverse effect of decreasing the ion source temperature for 4TMS-BaPT is an increase in asymmetry

7 120 % 284 100 U44V. (a) 50

266 285 89 255 446 355 0 m/z -20 50 100 150 200 250 300 350 400 450 500 550 600 650 120 % 284 100

446 FH4V. (b) 50 355 266 447 285 89 356 267 428 448 0 m/z -20 50 100 150 200 250 300 350 400 450 500 550 600 650 120 % 446 100 F44V.

355 50 (c) 428 447 266 89 356 429 448 267 357 0 m/z -20 50 100 150 200 250 300 350 400 450 500 550 600 650

Figure 8. Comparison of mass spectra for 4TMS-BaPT at varying ion source temperatures (a) 300 °C, (b) 250 °C and (c) 200 °C

120 % 620 100 492

75 200°C 472

50 (a)

147 493 621 25 383 452

0 m/z -20 50 100 200 300 400 500 600 700 120 % 620 100 175°C 75

472 50 (b)

621 25 147 473 383 640 0 m/z -20 50 100 200 300 400 500 600 700 120 % 620 100 150°C 75

50 472 (c)

492 621 25 147 473 640 0 m/z -20 50 100 200 300 400 500 600 700 Figure 9. Comparison of mass spectra for PFPA-HFIP-derivatized 11-nor-9-Carboxy-Δ9-THC at varying ion source temperatures (a) 200 °C, (b) 175 °C and (c) 150 °C 8 8.9e7 counts 4TMS-BaPT - RT=16.269; Peak Asymmetry=1.63 7.5e7

5.0e7 (a)

2.5e7 (a)

0.0e0 min -8.7e6

9.0e7 counts 8.0e7 4TMS-BaPT - RT=16.268; Peak Asymmetry=2.46

6.0e7 (b) 4.0e7

2.0e7 (b)

min 0.0e0

8.3e7 counts

6.0e7 4TMS-BaPT - RT=16.272; Peak Asymmetry=4.09 (c) 4.0e7 (c) 2.0e7

min 4.7e5 15.547 15.625 15.750 15.875 16.000 16.125 16.250 16.375 16.500 16.625 16.750 16.875 16.975

Figure 10. Chromatographic peak shape comparisons for TMS derivatized BaPT at varying ion source temperatures (a) 300 °C, (b) 250 °C and (c) 200 °C

8.9e7 counts 11norTHCCOOH - RT: 6.35; Peak Asymmetry:1.35 7.5e7

5.0e7 (a) (a) 2.5e7

0.0e0 min -1.2e7

1.0e8 counts 11norTHCCOOH - RT: 6.35; Peak Asymmetry:1.72

7.5e7

5.0e7 (b)

2.5e7 (b) 0.0e0 min -1.0e7

8.0e7 counts 11norTHCCOOH - RT: 6.35; Peak Asymmetry:1.76 6.0e7

4.0e7 (c)

2.0e7 (c) 0.0e0 min -1.0e7 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 6.95

Figure 11. Chromatographic peak shape comparisons for PFPA-HFIP-derivatized 11-nor-9-Carboxy-Δ9-THC at varying ion source temperatures (a) 200 °C, (b) 175 °C and (c) 150 °C

9 The effect of methane reagent gas flow rate on mass Conclusion ratios for 4TMS-BaPT at ion source temperature of In conclusion, this technical note illustrates the NCI 300 °C is shown in Figure 12. Increasing the methane capabilities of the ISQ 7000 single quadrupole GC-MS flow rate results in a higher degree of undesired system, with detection limits as low as 100 attograms fragmentation. This could be attributed to the greater OFN on-column. An appropriate balance of ion source abundance of thermal electrons resulting from the higher temperature and reagent gas flow rate is required to flow rate of methane, which in turn results in greater have a suitable abundance of high mass ions as well as dissociative electron capture reactions. An extreme case satisfactory peak asymmetry. For applications involving of high ion source temperature and high methane flow silylated compounds careful consideration needs to be rate is shown in Figure 13. Ion source temperature of given to optimization of source temperature, in order to 350 °C and a methane flow rate of 3 mL/min results in avoid excessive fragmentation of higher mass ions and almost complete disappearance of mass 446; the peak excessive peak tailing. For most applications however, asymmetry for this example was 1.0. ion source temperatures between 200 and 250 °C, and methane flow rates between 1 and 2 mL/min provide a 0.14 good balance between sensitivity and robustness.

0.12 References 0.1 1. Rosenfelder, N.; Vetter, W. Gas chromatography coupled to electron capture negative ion mass spectrometry with nitrogen as the reagent gas – an alternative method for 0.08 determination of polybrominated compounds. Rapid Commun. Mass Spectrom. 2009, 23, 3807-3812. 0.06 2. Tagami, T.; Kajimura, K.; Takagi, S.; Satsuki, Y.; Nakamura, A.; Okihashi, M.; Akutsu, Ratio of 446 to 284 0.04 M.; Obana, H.; Kitagawa, M. Simultaneous analysis of 17 organochlorine pesticides in natural medicines by GC/MS with negative chemical ionization. Yakugaku Zasshi: 0.02 Journal of the Pharmaceutical society of Japan 2007, 127(7), 1167-1171. 3. Xiao, X-Y.; McCalley, D.; McEvoy, J. Analysis of estrogens in river water and effluents 0 1 1.5 2 2.5 3 3.5 using solid-phase extraction and gas chromatography-negative chemical ionization Methane flow rate, mL/min mass spectrometry of the pentafluorobenzoyl derivatives. J. Chromatogr. A 2001, 923, 195-204. Figure 12. Effect of reagent methane gas flow rate on ratio of 4. Sparkman, O. D.; Penton, Z. E.; Kitson, F. G. Gas Chromatography and Mass m/z 446 to m/z 284 Spectrometry – A Practical Guide; Academic Press, 2011. 5. Day, B.; Naylor, S.; Gan, L-S; Sahali, Y.; Nguyen, T.; Skipper, P.; Wishnok, J.; 120 Tannenbaum, S. Molecular Dosimetry of Polycyclic Aromatic Hydrocarbon Epoxides % and Diol Epoxides via Hemoglobin Adducts Cancer Research 1990, 50, 4611-4618. 284 100 6. Huestis, M., A.; Barnes, A.; Smith, M. L. Estimating the Time of Last Cannabis Use from

80 Plasma Δ9 -Tetrahydrocannabinol and 11-nor-9- Carboxy-Δ9 -Tetrahydrocannabinol Concentrations. Clinical Chemistry 2005, 51-12, 2289-2295. 60 7. Ciceri, E.; Albertini, T; Caruso, A. Sensitive determination of THCCOOH in hair to 89 regulatory requirements using Triple Quadrupole GC-MS/MS. AN 10493, Thermo 40 266 255 Fisher Scientific. 285 20 8. Harrison, A. G. Chemical Ionization Mass Spectrometry; CRC press, 1992. 267 240 355 9. McLafferty, F. W.; Turecˇek, F. Interpretation of Mass Spectra; University Science Books, 0 1993.

m/z -20 10. Gujar, A.; Anderson, T.; Cavagnino, D.; Patel, A. Practical determination and validation 50 100 200 300 400 500 600 650 of instrument detection limit for the Thermo Scientific ISQ 7000 Single Quadrupole GC-MS system with Advanced Electron Ionization source. TN 10597, Thermo Fisher Figure 13. Mass spectrum of 4TMS-BaPT at 350 °C and 3 mL/min Scientific. methane gas flow rate

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