Low-level 40Ca determinations using nitrous oxide with reaction cell inductively coupled plasma-tandem mass spectrometry.

In inductively coupled plasma mass spectrometry, the most abundant Ca isotope (40Ca) suffers from isobaric interference with argon, hindering the potential for low detection limits of Ca. A powerful approach is to remove the interference by using a reaction gas in a reaction cell. Ammonia (NH3) has proven to be an effective reaction gas by process of a charge transfer reaction. However, NH3 is highly corrosive and toxic and cannot remove isobaric 40 K. Therefore, this work proposes the use of nitrous oxide (N2O) to mass shift the target analyte 40Ca to 40Ca16O+ as a non-corrosive and non-toxic alternative. Instrument performance testing demonstrated that N2O was capable of reaching equivalent detection limits (0.015 ng g-1) and background equivalence concentrations (0.041 ng g-1) to that of NH3 and limited by the blank only. Further investigation of matrix interferences with synthetic standards highlighted that the N2O approach supports the separation of potassium (K) and magnesium (Mg)-based interferences at tested concentrations of more than 600 times and almost 800 times higher than Ca respectively, whereas NH3 was found to only support the removal of Mg. This work highlights a clear advantage of N2O for low-level Ca determinations with high matrix loads, as well as compatibility with other instrumentation sensitive to corrosion that supports reaction cell technology.

Introduction

Inductively coupled plasma mass spectrometry (ICP-MS) is a widely used tool that boasts high sensitivity, low limit of detection (LOD), and high sample throughput. However, LODs of ICP-MS measurements can suffer due to spectral interferences from monatomic or polyatomic ions with the same mass to charge ratio (m/z). Calcium (Ca) is an example of an element that suffers from such interferences. In this case, the major isotope, 40Ca, which has a natural abundance of 96.941% [1], shares an isobaric interference with argon (Ar), which is used as both a carrier gas and for the generation of the plasma. To separate these, the required mass resolution would be > 190,000 (Table 1). As such, sensitivity and detection limits are greatly hampered by resorting to using less abundant Ca isotopes, such as 42Ca (0.647% abundance), 43Ca (0.135% abundance), and 44Ca (2.086% abundance) [1].

Table 1

List of interferences for Ca on m/z 40. Mass resolution was calculated from IUPAC Periodic Table of the Elements and Isotopes [1]. Negative resolutions indicate that the atomic mass of the interfering species is greater than that of.40Ca

Interference on 40Ca+	Abundance (%)	Atomic mass (u)	Required mass resolution
40Ca+	96.9	39.943	–
40Ar+	99.6	39.962	192,058
40 K+	0.0117	39.964	− 28,394
24Mg16O+	78.8	39.980	− 2302
80Se++	49.6	39.958	9229
80Kr++	2.29	39.958	9079
39K1H+	93.2	39.972	− 4471
23Na17O+	0.038	39.989	− 1520

One method commonly employed to overcome spectral interferences is the introduction of a reaction gas via a reaction cell. Recently developed instruments utilize an additional quadrupole as a mass filter in front of the reaction cell to avoid the formation of new interferences in the cell (ICP–tandem mass spectrometry (MS/MS)) [2, 3]. Ammonia (NH3) has been typically used as a reaction gas to remove interference of 40Ar+ for on-mass determination of 40Ca+ [4], as the charge transfer reaction (M+  + NH3 → NH3+  + M) occurs at a much higher rate for Ar+ than for Ca+ [2, 5, 6]. However, NH3 is a corrosive and toxic gas [7]. Therefore, it cannot be used in every instrument and its usage is sometimes limited in some laboratories. It is therefore of interest to assess suitable alternatives for greater sustainability regarding primarily the protection of the instrumentation.

Other reaction gases have also been used for on-mass determinations of 40Ca, such as methane [8, 9] and hydrogen [10]. However, mass-shift reactions of 40Ca using a reaction gas (e.g. 40Ca → 40Ca16O+) are less widely reported. Oxygen is the traditional reaction gas for analyte mass-shift determinations [2]. However, low formation of the CaO+ product ion renders this approach unfavourable [11]. Nitrous oxide (N2O), on the other hand, is a more reactive alternative to oxygen [12, 13] and has been explored recently for a number of elements using ICP-MS/MS systems [14-16], highlighting a broad scope for use in routine multi-element analysis. While N2O shows high reactivity, it is notably much less corrosive than NH3 and may serve as a suitable alternative. Apart from 40Ar, other interferences have to be considered if Ca is determined at low levels in a complex matrix (see Table 1).

While sensitivity can be enhanced and the LOD can be decreased by using cell methodology, it is important to note that current LODs are limited by background levels of Ca. Therefore, it is important to be highly considerate of sources of contamination. Wu et al. suggested the use of a clean laboratory environment can allow for lower detection limits [9]. Retzmann et al. described in detail how to minimize the Ca background and reported that, e.g., the use of nitrile gloves and clean-room wipes were major sources of Ca contamination and should be avoided [17].

This work aims to evaluate the novel usage of N2O for quantification of Ca at low levels in complex matrices by ICP-MS/MS using 40Ca. Optimization of both the cell gas flow rates and possible internal standards was carried out. Instrument performance parameters for both reaction gases (NH3 and N2O) were compared with each other, as well as with the determination of 40Ca under standard conditions (no cell gas). Additionally, the effectiveness of the removal of sample matrix interferences caused mainly by magnesium (Mg) and potassium (K) was also investigated for each cell gas.

Materials and methods

All preparations and measurements were made in a clean room (ISO class 8) to minimize the risks of contamination. Polyethylene gloves (Carl Roth GmbH, Karlsruhe, Germany) were used on top of nitrile gloves to avoid Ca contamination. The use of clean-room wipes and paper towels were avoided throughout.

Chemicals and standards

Nitric acid (w = 65%, p.a. grade; Carl Roth GmbH) was first purified by sub-boiling using a sub-boiling distillation system (Savillex DST-4000, AHF Analysentechnik, Tübingen, Germany). Reagent grade I water (18.2 MΩ cm; MilliQ IQ 7000, Merck-Millipore, Darmstadt, Germany) was used for all acid dilutions. Sample vials and pipette tips were pre-cleaned by soaking overnight in diluted sub-boiled nitric acid (w = 10% and subsequently w = 3% respectively) before use.

All standards were prepared in dilute sub-boiled acid (w = 2%). A Ca single element ICP-MS standard (β = 1000 µg mL−1; CertiPur, Merck, Darmstadt, Germany) was used as stock for calibration preparations. Single element standards of scandium (Sc, β = 1000 µg mL−1; Inorganic Ventures, Christiansburg, VA, USA), yttrium (Y, β = 10 µg mL−1; Elemental Scientific, Omaha, NE, USA), and indium (In, β = 1000 µg mL−1; CertiPur, Merck) were used to produce a mixed internal standard. Additional 1000 µg mL−1 single element ICP-MS standards of magnesium (β = 1000 µg mL−1; CertiPur, Merck) and potassium (β = 1000 µg mL−1; CertiPur, Merck) were used for interference testing.

ICP-MS/MS measurements

All ICP-MS/MS measurements were carried out using a NexION 5000 (Perkin Elmer, Waltham, MA, USA), which is equipped with a dynamic reaction cell (DRC). Instrumental parameters are listed in Table 2. The sample was introduced to the ICP via a peristaltic pump. A mixed internal standard containing Sc (w = 4.7 ng g−1), Y (w = 2.2 ng g−1), and In (w = 2.2 ng g−1) was added online. Measurement parameters for the internal standards are displayed in Table 3. Calibrations in standard mode (both using single-quadrupole mode (Q3) and MS/MS mode) were carried out in the range of 5–1000 ng g−1 Ca and measured using the 44Ca isotope. DRC calibrations were carried out in the range of 0.2–100 ng g−1 Ca and measured using both the 40Ca and 44Ca isotopes. Using N2O, Ca was detected as the 40Ca16O+ and 44Ca16O+ product ions (mass shift of +16 amu).

Table 2

Instrument and plasma conditions for measurements made using ICP-MS/MS

Parameter	Q3	MS/MS	NH3 DRCMS/MS	N2O DRCMS/MS
Measurement mode	Standard	Standard	DRC on mass	DRC mass shift
Cell gas	None	None	NH3	N2O
Cell gas flow rate	None	None	0.7 mL min−1	0.4 mL min−1
RPa	0	0	0	0
RPq	0.25	0.25	0.45	0.45
Sample introduction	Peristaltic pump	Peristaltic pump	Peristaltic pump	Peristaltic pump
Nebulizer	PFA MicroFlow	PFA MicroFlow	PFA MicroFlow	PFA MicroFlow
Spray chamber	Peltier cooled SilQ cyclonic spray chamber	Peltier cooled SilQ cyclonic spray chamber	Peltier cooled SilQ cyclonic spray chamber	Peltier cooled SilQ cyclonic spray chamber
Spray chamber temperature	5 °C	5 °C	5 °C	5 °C
Interface cones	Nickel	Nickel	Nickel	Nickel
RF power	1600 W	1600 W	1600 W	1600 W
Ar nebulizer gas flow	0.98 L min−1	0.98 L min−1	0.98 L min−1	0.98 L min−1
Ar auxiliary gas flow	1.2 L min−1	1.2 L min−1	1.2 L min−1	1.2 L min−1
Ar plasma gas flow	16 L min−1	16 L min−1	16 L min−1	16 L min−1
QID fixed voltage	− 12 V	− 12 V	− 12 V	− 12 V
Hyperskimmer park voltage	5 V	5 V	5 V	5 V
OmniRing park voltage	− 185 V	− 185 V	− 185 V	− 185 V
Inner target lens voltage	2 V	2 V	2 V	2 V
Outer target lens voltage	− 7 V	− 7 V	− 7 V	− 7 V
Deflector exit voltage	− 8 V	− 8 V	− 8 V	− 8 V
Differential aperture voltage	− 3.5 V	− 3.5 V	− 3.5 V	− 3.5 V
Q1 AC rod offset	− 7 V	− 6 V	− 5 V	− 7.5 V
Q1 rod offset	− 11 V	− 2 V	− 0 V	0 V
Cell rod offset	− 33 V	− 33 V	− 5 V	− 2 V
Axial field voltage	0 V	0 V	150 V	250 V
Cell entrance voltage	− 10 V	− 5 V	− 6.5 V	− 7.5 V
Cell exit voltage	− 2 V	− 2 V	− 7 V	− 5 V
Q3 AC rod offset	− 2.5 V	− 2.5 V	− 8.5 V	− 8 V
Q3 rod offset	− 2 V	− 2 V	− 13 V	− 10 V
Dwell time	50 ms	50 ms	50 ms	50 ms
Scans	6	6	6	6
Replicates	6	6	6	6

Table 3

ICP-MS/MS measurement conditions for the tested internal standards using N2O and NH3 DRC

Internal standard	Reaction gas	Q1m/z	Q3m/z
Sc	N2O	45	61
Y	N2O	89	105
In	N2O	115	115
Sc	NH3	45	45
Y	NH3	89	89
In	NH3	115	115

Cell gas flow rate optimization

Optimization of the reaction gas flow rates for NH3 and N2O was carried out on m/z 40 using a 10-ng g−1 single element Ca standard and a blank solution (w = 2% HNO3). Tested flow rates ranged from 0.1 to 1.0 mL min−1 using N2O and from 0.4 to 1.5 mL min−1 using NH3.

Evaluation of matrix interferences

Evaluation of potential interference caused by Mg and K using N2O and NH3 DRC was carried out by measurement of the Ca background equivalence concentration in each respective single element standard at dilutions between 0.5 and 5.0 µg g−1. Determined Ca concentrations, made by means of external calibration, were used to evaluate the significance of each interference by comparing the mass fraction of Ca obtained using the non-interfered (by isobaric interference) isotope on m/z 44 (w(Ca) 44) to that of the interfered isotope on m/z 40 (w(Ca) 40). Where the difference was found to be significant, the magnitude of interference was calculated by subtraction of w(Ca) 44 from w(Ca) 40.

Certified reference materials

River water certified reference materials (CRMs), SLRS-3 and SLRS-5 (both National Research Council Canada, Ontario, Canada), were used for validation. The materials were diluted to approximately 1 ng mL−1 Ca using dilute sub-boiled acid (w = 2% HNO3). Analysis was carried out using N2O and NH3 as reaction gases.

Statistics

Statistical testing and the generation of figures were carried out using RStudio (version 2021.9.2.382). LOD and LOQ were calculated respectively from 3 and 10 times the standard deviation of 5 repeated measurements of a blank solution (w = 2% HNO3). Background equivalence concentration (BEC) was calculated from the calibration by division of the background (y-intercept) with the calibration slope. Equidistant standard concentrations of Ca between 0.2 and 1.0 ng g−1 were used when determining the LOD, LOQ, and BEC of the DRC methods.

Evaluation of the effect of interferences was carried out using total least squares (Deming) regression on the concentrations obtained using the 40Ca and 44Ca isotopes, where standard errors were calculated using the jackknife method. The observed w(Ca) 40/w(Ca) 44 ratio was statistically compared to the expected ratio of 1 using a Z test.

Results and discussion

The profile observed for mass shift using N2O (Fig. 1A) displayed optimum sensitivity for the formation of the 40Ca16O+ product ion at 0.4 mL min−1. The background signal of 40Ar+ was found to decrease as the N2O flow rate increased. Further observations of the blank signal conducted using the 44Ca isotope at optimum N2O conditions found a similar signal ratio of 40Ca/44Ca to that of the natural abundance. This suggests that, while a small degree of 40Ar16O+ forms at lower N2O flow rates, Ar was successfully removed by N2O at optimum conditions and the observed blank signal is primarily due to the presence of background Ca.

Fig. 1

Optimization of sensitivity for 10 ng g−1 Ca (solid line) using A N2O and B NH3 cell gas flow rate for the removal of 40Ar interference on.40Ca. The background signal, measured using a blank solution, is also indicated (dotted line). Variation in the signal to background ratio (SBR) for NH3 DRC is shown in C. Error bars present represent one standard deviation of six replicates

On-mass determination of Ca was carried out using NH3 flow rates between 0.4 and 1.5 mL min−1, as preliminary tests (carried out using 38Ar) suggested that lower flow rates would not remove enough Ar to prevent detector saturation on m/z 40. Within this range, the signal for Ca was observed to decrease with increasing NH3 flow (Fig. 1B). Consideration of the signal to background ratio demonstrated a plateau at NH3 flow rates greater than 0.7 mL min−1 (Fig. 1C); hence, this was determined to be the optimum condition. Similar to the use of N2O DRC, additional monitoring of the blank using 44Ca highlighted that the blank signal obtained on m/z 40 at the optimum NH3 flow rate was due to the presence of background Ca.

Internal standards

Sc, Y, and In were tested as possible internal standards, as they have a narrow range of ionization energies (5.78–6.56 eV) close to that of Ca (6.11 eV) [18]. Variation of the internal standard response to the DRC gas flow rate is shown in Fig. 2. Initial tests using N2O DRC highlighted that In could not be measured in mass-shift mode, as no formation of the InO+ product ion was observed. However, detection of In+ on mass with N2O was found to be feasible, as an adequate and stable signal was obtained. Given that the sensitivity of the In signal increased with N2O flow rates up to 0.7 mL min−1, it could be interpreted that the signal observed may be enhanced by collisional focussing. Sc and Y, on the other hand, were observed to form the oxide product ion. Additionally, the sensitivity profiles closely matched that of Ca, with both internal standards displaying an optimum N2O flow rate of 0.4 mL min−1. The use of Sc and Y may then prove more advantageous in comparison to In, as they display similar cell reaction characteristics to that of the analyte.

Fig. 2

Variation of 45Sc (solid line), 89Y (dashed line), and.115In (dotted line) internal standard signal with A N2O and B NH3 cell gas flow rate. Error bars present represent one standard deviation of six replicates

In contrast, Sc and Y were observed to be unfit for use as internal standards when using NH3 as a reaction gas (Fig. 2B), as both elements were effectively removed when measuring on mass. The profile of In showed a collisional focussing effect, where a sufficient and stable signal was obtained for use as an internal standard at the previously determined optimum NH3 flow rate of 0.7 mL min−1. Given that, of the three internal standards considered, In was the only one found to be suitable for measurements, all subsequent data reported for NH3 DRC only includes internal normalization to In.

Performance data

The instrument performance for the two DRC methods, as well as standard mode, was assessed and is presented in Table 4. Between the three internal standards used for N2O DRC, minimal differences in sensitivity (slope = 128,000–129,000 cps/ng g−1) and BEC (0.41–0.42 ng g−1) were observed. However, stability data indicated a lower RSD when using Sc (1.6%) as an internal standard compared to Y (2.2%) and In (2.9%). This was also reflected in the slightly lower LOD (of 0.015 ng g−1) and LOQ (of 0.049 ng g−1) values obtained using Sc. Therefore, while all three internal standards can be used for determination of Ca, Sc has been shown here to be the optimal in this case.

Table 4

Comparison of calibration parameters for the measurement of 44Ca with no reaction gas in Q3 and MS/MS mode, and.40Ca in MS/MS mode with N2O and NH3 reaction gas

Parameter	44CaQ3 mode(no cell gas)	44CaMS/MS mode(no cell gas)	40Caon massNH3 DRC	40Camass shiftN2O DRC	40Camass shiftN2O DRC	40Camass shiftN2O DRC
Internal standard	Indium	Indium	Indium	Scandium	Yttrium	Indium
Slope (cps/ng g−1)	8260	3650	226,000	129,000	129,000	128,000
Intercept (cps)	150,000	56,500	82,500	52,600	52,700	53,300
Correlation, R	0.99983	0.99968	0.99832	0.99941	0.99924	0.99930
LOD (ng g−1)	0.14	0.27	0.015	0.015	0.017	0.017
LOQ (ng g−1)	0.43	0.81	0.049	0.049	0.057	0.056
BEC (ng g−1)	18	15	0.37	0.41	0.41	0.42
Stability test accuracy (%)*	–	–	101	101	99.3	97.0
Stability test RSD (%)*	–	–	2.39	1.57	2.20	2.92

*Based on 20 replicate measurements of a 0.5-ng g.−1 Ca standard

While the calibration slope observed for 40Ca using NH3 was 1.75 times greater than that of the N2O, the LOD (of 0.015 ng g−1) and LOQ (of 0.049 ng g−1) remained consistent. Additionally, determined BEC values (of 0.37 ng g−1 and 0.41 ng g−1 for NH3 and N2O DRC respectively) were similar between the two methods. This further indicates that the background signal for both DRC methods is likely due to background Ca levels. In comparison to the measurement of 44Ca using the Q3 mode with no cell gas (which is more sensitive than the MS/MS mode with no cell gas), the application of both NH3 and N2O DRC methods showed marked improvement, with approximately 10 times lower LOD (of 0.015 ng g−1) and LOQ (of 0.049 ng g−1), and approximately 45 times lower BEC (of 0.42 ng g−1).

Removal of interferences

Mg and K were identified as the primary interferences of concern for measurements using the 40Ca isotope, as the polyatomic 24Mg16O+ interference formed in the plasma and isobaric interference from 40 K+ cannot be removed by the Q1 mass filter. Selenium, as 80Se++, was not considered to be of concern due to the high ionization energy required to form such interfering species [18] (Table 5).

Table 5

Total least squares regression results for the comparison of determined background Ca concentrations (w = 0–7.5 ng g−1) using isotopes 44Ca and 40Ca in high-concentration Mg (w = 0–4.0 µg g−1) and K (w = 0–4.4 µg g.−1) single element standards

DRC mode	Slopew(Ca)m/z 40 vs. w(Ca)m/z 44	Standard error of the slope	Z testp value	Significant?	Lower confidence interval	Upper confidence interval
Potassium interference
N2O at 0.4 mL min−1 (Sc Int. Std.)	0.9917	0.0062	> 0.150	No	0.96518	1.01825
N2O at 0.4 mL min−1 (Y Int. Std.)	0.9916	0.0061	> 0.150	No	0.96532	1.01797
N2O at 0.4 mL min−1 (In Int. Std.)	0.9915	0.0061	> 0.150	No	0.96526	1.01777
NH3 at 0.7 mL min−1 (In Int. Std.)	1.1335	0.0022	< 0.001	Yes	1.12386	1.14314
Magnesium interference
N2O at 0.4 mL min−1 (Sc Int. Std.)	0.9982	0.0107	> 0.150	No	0.95212	1.04429
N2O at 0.4 mL min−1 (Y Int. Std.)	0.9986	0.0111	> 0.150	No	0.95084	1.04626
N2O at 0.4 mL min−1 (In Int. Std.)	0.9987	0.0104	> 0.150	No	0.95399	1.04336
NH3 at 0.7 mL min−1 (In Int. Std.)	1.0062	0.0128	> 0.150	No	0.95135	1.06103

Native Ca concentrations in the measured single element standards of both Mg and K ranged between 1 and 10 ng g−1. For measurements of Ca with high Mg load, the w(Ca) 40/w(Ca) 44 ratio did not significantly differ from 1 for both N2O and NH3, which indicates that both gases can achieve successful removal of 24Mg16O+ interference on m/z 40. However, measurements of native Ca in high-level K standards using NH3 DRC showed a significantly greater w(Ca) 40/w(Ca) 44 ratio than expected, indicating significant bias introduced due to the presence of interfering 40 K that was not removed. Conversely, no such significant bias was observed for measurements of K single element standards using N2O DRC, suggesting that N2O can be used to successfully overcome the interference of K.

The level of interference from 40 K using NH3 DRC was determined by the difference of the observed w(Ca) 40 and w(Ca) 44 and plotted against the prepared K concentration (Fig. 3). The slope of the plot indicated that the level of interference on 40Ca appears as 0.0128% of the matrix K concentration. To obtain < 1% interference on 40Ca, matrix K concentrations cannot exceed about 80 times that of the analyte Ca concentrations. Further variation of the NH3 flow rate was not able to overcome the isobaric interference from K, as the Ca/K signal ratio only decreased with increasing flow rate (Fig. 4).

Fig. 3

Contribution of 40 K interference on 40Ca measurements of single element K standards (w = 0–4.4 µg g.−1) using NH3 DRC. Error bars present represent the combined standard deviation of the difference between the observed w(Ca) 40 and w(Ca) 44 (calculated by the law of propagation of uncertainties)

Fig. 4

Variation of the relative signal to background ratio (SBR) with NH3 flow rate (normalized to the SBR observed at 0.7 mL min−1 NH3) for 10 ng g−1 40Ca signal with a 25 ng g−1.39 K background (solid line) and blank background on m/z 40 (dotted line). Error bars represent one combined standard deviation of six replicates (calculated by the law of propagation of uncertainties)

Method validation

Validation of the total determination of Ca using N2O DRC was carried out using two river water CRMs that were certified for Ca. Results are shown in Table 6. Excellent recoveries between 99.2 and 103% were obtained for the two CRMs using Sc and Y as internal standards. Slightly lower recoveries of 95.4–96.9% were obtained using In as an internal standard, though these still fall within the stated uncertainty of the CRM. Recoveries of 97.9–98.5% were obtained using NH3 DRC, indicating that the N2O DRC method for determination of total Ca performs as well as existing methodology for real sample matrices.

Table 6

Measured mass fractions and recoveries of river water CRMs (diluted to 1 ng mL.−1) using N2O and NH3 DRC

CRM	Certified mass fraction (µg g−1)	N2O DRC (Sc internal standard)		N2O DRC (Y internal standard)		N2O DRC (In internal standard)		NH3 DRC (In internal standard)
		Measured (µg g−1)	Recovery (%)	Measured (µg g−1)	Recovery (%)	Measured (µg g−1)	Recovery (%)	Measured (µg g−1)	Recovery (%)
SLRS-3	6.0 ± 0.4	6.09 ± 0.09	101	5.95 ± 0.10	99.2	5.72 ± 0.09	95.4	5.87 ± 0.09	97.9
SLRS-5	10.5 ± 0.4	10.8 ± 0.1	103	10.8 ± 0.1	103	10.2 ± 0.1	96.9	10.3 ± 0.1	98.5

Conclusions

The data presented within this study suggests that N2O is not only a suitable replacement for NH3 for total Ca determinations, but can also ensure matrix-free determinations of 40Ca, especially in K-rich matrices with low Ca content. Despite apparent lower sensitivity of the N2O approach (by a factor of 1.75), detection limits and BEC were found to be similar between the two cell gases. We therefore propose that N2O should be used in place of NH3 for routine measurements of Ca, as well as incorporating this approach into wider multi-element analysis.