Determination of selenium in serum in the presence of gadolinium with ICP-QQQ-MS

Graphical Abstract Triple-quadrupole ICP-MS using O 2 mass-shift technology is superior for removing gadolinium interference on selenium in serum. Gadolinium (Gd)-based magnetic resonance imaging (MRI) contrasting agents interfere with the determination of Se when analysed by single quadrupole inductively coupled plasma-mass spectrometry (ICP-MS). This paper demonstrates that an ICP-triple quadrupole-MS (ICP-QQQ-MS) with oxygen mass shift overcomes Gd ++ interference on Se + and mitigates typically encountered matrix and spectral based interferences. Normal human serum was diluted in a solution containing isopropanol, EDTA, NH 4 OH and Triton X-100. Samples were unspiked (control) serum; serum spiked with 0.127 µmol L -1 Se or 127 µmol L -1 Gd; and serum spiked with both 0.127 µmol L -1 Se and 127 µmol L -1 Gd. Consideration of collision/reaction gases and conditions for interference mitigation included helium (He); a ‘low’ and ‘high’ hydrogen (H 2 ) flow, and oxygen (O 2 ). The instrument tune for O 2 was optimised for effective elimination of interferences via a mass shift reaction of Se + to SeO + . The ICP-QQQ-MS was capable of detecting trace (> 9.34 nmol L -1 ) levels of Se in serum in the presence of Gd in our simulated post-MRI serum sample. The multi-tune capabilities of the ICP-QQQ-MS may be adapted to eliminate other specific isobaric interferences


Introduction
Selenium (Se) is an essential trace element that is incorporated into the amino acid backbone of proteins as selenocysteine. Se has structural and enzymatic roles in 25 known human selenoproteins. [1][2][3] These proteins are antioxidants; involved in the production of active thyroid hormone; roles in immune function; control of viruses such as HIV; prevention of cancer; 4 and are important for reproductive health. [4][5][6][7][8][9] Selenium deficiency has been associated with diseases affecting nutrient absorption including coeliac disease, some types of cancer, restricted diets and Keshan disease. 10-12 ; 4,13,14 Additionally, a significant decline in blood Se levels with age has been reported. 15 . Stated serum levels vary geographically. For example, in healthy European populations levels ranged from around 0.5 to 1.2 µmol L -1 of Se. [15][16][17] while levels as low as 0.34 µmol L -1 have been reported in populations where Keshan disease occurs. 18 Single quadrupole inductively coupled plasma-mass spectrometry (SQ-ICP-MS) is the preferred standard analytical method for analysis of most elements in biological fluids, and represents 95% of all commercial ICP-MS instruments. 19 The two most abundant isotopes are 80 Se and 78 Se, with 82 Se the preferred isotope pre-collision cell technology along with interference equation correction, due to less isobaric interferences from the argon plasma and the sample matrix (Table 1), whilst 78 Se is the preferred isotope when using collision cells. Gadolinium (Gd) is widely used in contrasting agents for magnetic resonance imaging (MRI) of the vasculature and tumours of the central nervous system, 20 and interferes with the major isotopes of Se due to doubly charged Gd species. [21][22][23] Consequently, incorrect diagnosis of Se toxicity may arise from the presence of high levels of circulating Gd. Total elimination of circulating Gd may be in the order of days or longer for those with impaired renal function. 24 For example, in 2008, a clinical urine sample measured by ICP-MS returned a Se concentration of 16.8 µmol L -1 , a level associated with acute Se poisoning. This measurement was an artefact resulting from an MRI procedure performed on the day of testing using a Gd contrasting agent. 22 Interference on Se is typically managed by a collision/reaction cell which removes polyatomic species with kinetic energy discrimination or by chemically induced dissociation, providing reduced backgrounds and improved limits of detection and quantification. 21,25 The higher mass resolving power of double-focusing sector field massspectrometers (ICP-SF-MS) is an alternative to reducing the impact of polyatomic and isobaric interferences on mass measurements. [26][27][28] However, ICP-SF-MS are costly, complex and high resolution settings compromise the sensitivity of the analysis when resolving Ar dimers from Se. Comparisons of SQ-ICP-MS with a collision/reaction cell against a SF-ICP-MS demonstrated that 80 Se + could not be resolved from 40 Ar 2 + in the high-resolution SF-ICP-MS and the SQ-ICP-MS had superior isotope ratio precision and a lower LOD. 30 Mathematical corrections may also be used to remove the signal from doubly charged 31 and polyatomic species, 32 but are less accurate than removing interfering species by physical methods. The triple quadrupole ICP-MS (ICP-QQQ-MS) reduces interferences by operating in either standard single quadrupole (SQ) mode or tandem MS/MS. In the MS/MS mode a quadrupole (Q1) filters the mass-to-charge ratio (m/z) of interest prior to introduction into an ion-guide (Q2), which has the option to be filled with a collision and/or reaction gas. The final quadruple (Q3) again filters the desired analyte, either on its original mass or the known reaction product. 33 O 2 may be used to react with kinetically favoured analytes to an MO + mass filtered by Q3, removing all other interfering species. This approach was applied to the measurement of selenoproteins in rat serum by liquid chromatography (LC)-ICP-MS/MS 34 and arsenic (As) and Se in food. 35 This MO + mass shift improved detection limits for phosphorus (P), sulfur (S) and silicon (Si) over those obtained from isotope dilution (ID)-ICP-SF-MS in both aqueous 34 and organic matrices. 34,36 O 2 reaction might produce unwanted polyatomic species, then alternative reaction / collision gases may be used, e.g. NH 3 /He for the ultra-trace detection of titanium (Ti; as Ti(NH 3 ) 6 + ) in biological fluids. 37 This paper describes the analysis of clinically relevant concentrations of Se in serum samples in the presence of Gd via an O 2 mass shift approach.

Data analysis
Following instrument calibration by standard addition, sample concentrations for each m/z in the 10 replicate samples was calculated using the Agilent ICP-MS MassHunter data analysis software. Statistical analysis (Student's t-test, p < 0.05) was performed in Prism 6 (GraphPad, La Jolla, California, United States of America).

Collision/reaction conditions
Four tune modes were examined for the determination of Se in human serum under conditions simulating those following the administration of a Gd MRI contrasting agent. The modes represented three commonly used methods of interference removal; kinetic energy discrimination (He), removal via reaction with hydrogen (H 2 ), or adduct formation via reaction with O 2 . For tune mode 1, Q1 was set as an ion guide. Tune 1 (HE-He) used a high potential difference (the OctP bias) to accelerate incoming ions into the He-filled octopole cell for collisional induced dissociation of the ArAr dimer. Energy discrimination was optimised to reduce ions escaping the confines of the cell maintaining a low background. For tunes 2 and 3 (H 2 tunes) the cell was optimised for reaction mode. To Table 3 Concentration of Se (± 1 standard deviation) in samples per tune mode (µmol L -1 ).  facilitate desirable in-cell reactions, a low negative voltage OctP bias accelerated ions into the cell, and a slight positive bias limited in-cell products from exiting the cell, providing more time for reaction. Tune mode 2 used an H 2 flow rate of 5.0 mL min -1 (standard conditions for Se determination, to reduce the 40 Ar 38 Ar + dimer at m/z 78), and tune mode 3 used a higher flow rate (9.0 mL min -1 ), determined experimentally as the optimal rate to reduce 156 Gd ++ interference on m/z 78, whilst maintaining adequate sensitivity for 78 Se + (Figure 1). In the case of tune mode 4 (O 2 mass shift), the energy and cell gas flow was optimised to maximise the low yield endothermic Se + + O → SeO + reaction (δ 0.69 eV), and to minimise the exothermic reaction of Gd ++ with O 2 (Gd + + O → GdO + , δ -2.39eV ). Energy discrimination of -5 V was experimentally determined to minimise 156 Gd 16 O 2 ++ and maximise 78 Se 16 O + passage through the collision/reaction cell and into Q3. The first mass filter (Q1) limited the transmission to the cell of plasma generated ions to the selected mass. The results are shown in Table 3.

Instrument performance
The amount of Se spiked into the serum standard was selected from typical ranges reported by Thomson. 41 The limits of detection (LOD) and quantification (LOQ) ( Table 4) were determined from the signal intensities using Equation 1, where y = 3 for LOD and 10 for LOQ.

Limit=
SD blank mean spikes − mean blanks × spike concentration

Equation 1
LOD and LOQ data were not acquired for 80 Se for tune modes 1-3 due to inability of the ICP-QQQ-MS to discriminate m/z 80 for 80 Se + and 40 Ar 2 + . Tune mode 4 permitted the monitoring of the 80 Se + isotope as 80 Se 16 O + .

Discussion
The isotopes of Se that may be determined are shown in Table  1. The most commonly measured isotope is 78 Se (23.8% NA), graphically represented in Figure 2a-d for each tune mode. The presence of the Gd ++ interference was not mitigated by the HE-  He tune (tune mode 1), the low H 2 tune (tune mode 2), or the high flow H 2 tune (tune mode 3), with Se concentrations overestimated by up to 20 times. The O 2 -induced mass shift using tune mode 4 sufficiently removed doubly-charged Gd interference, which also allowed measurement of the more highly-abundant 80 Se isotope by shifting the measured m/z away from the 40 Ar 2 + interference (Figure 2e). The O 2 mass shift (tune mode 4) was clearly the most accurate and precise method with recoveries of 99.7-101.8% for 77 Se, 78 Se, 80 Se and 82 Se from Se + Gd spiked serum samples.
Helium is often used as a collision gas to reduce interferences on the majority of elements. Tune mode 1 (HE-He) removed the ArAr interference on 77,78 Se + , however it did not eliminate the 154,156 Gd ++ signal. This tune did not remove the 40 Ar 40 Ar + interference on 80 Se + , whilst 82 Se + was not examined due to the high number of polyatomic interferences arising from the biological matrix (Table 1).
Tune mode 2 (low H 2 ) effectively removed the polyatomic interferences, but was unable to sufficiently reduce the interference caused by the Gd ++ ions. Tune mode 3 (high H 2 ) mitigated the polyatomic interferences on 77,78,82 Se + and eliminated the 154 Gd ++ (NA 2.18%) interference on 77 Se + (p < 0.05). Tune mode 3 failed to eliminate the 156 Gd ++ (NA 20.5%) interference on 78 Se + .This contrasts with Harrington et al. 21 who found that H 2 in the collision cell with a flow rate of 3.26 mL min -1 removed Gd ++ interference on the Se signal in serum due to the concentration of Se an order of magnitude higher than this study (0.127 µmol L -1 vs 1.01-3.56 µmol L -1 ). Similarly, Jackson et al. 35 removed the interference of 156 Gd ++ on the measurement of 78 Se + with a H 2 cell gas flow of 6 mL min -1 in food samples, also due to high to relatively high concentrations of Se; and a Gd spike 200 times lower than our simulation of post-MRI serum Gd concentration.
Other concerns with H 2 as a reaction gas include patients with high levels of circulating bromine, arising from bromhexine hydrochloride, a common ingredient in expectorants. H 2 reacts with 79 Br + and 81 Br + to form isobaric interferences on 80 Se + and 82 Se + , respectively. Deuterium has been used to overcome BrH + interferences, 42 though high expense limits its practical usage.
Tune mode 4 maximised the formation of Se 16 O + adducts and minimised the influence of doubly-charged Gd species on Se detection across all isotopes and was the superior method for Se determination, irrespective of isotope. Others have increased the yield of SeO + with mixed gases of H 2 and O 2 in the collision cell 35 . We also trialled mixed cell gases and did not observe any benefit.
Minimising unwanted masses entering, and preventing undesirable interactions, in the collision cell, is a significant feature of the ICP-QQQ-MS. For example S + may be removed from the ion path before it reaches the collision cell, where it could potentially form kinetically favouable species such as 32 S 16 O 3 + . The ICP-QQQ-MS may also find application in method validation strategies by ensuring isotopically pure signals particualarly for analytes known to be confounded by polyatomic or isobaric interferences.

Conclusions
Gd-based MRI contrasting agents interfere with Se analyses by ICP-MS. Reaction with O 2 using the ICP-QQQ-MS allowed a mass shift reaction of Se, which enabled detection of all major isotopes of Se with adequate sensitivity in the presence of Gd. Additionally, this approach overcame interferences from Ar dimers, further improving the sensitivity of the analysis. ICP-QQQ-MS has the unique capability to selectively isolate ions of interest from interferences or confounding signals at low concentrations, as demonstrated in this simulated scenario of Gd interference on serum Se levels.