Analytical Evaluation of Low-Field 31P NMR Spectroscopy for Lipid

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Analytical evaluation of low-field P NMR spectroscopy for lipid analysis Boris Gouilleux, Nichlas Vous Christensen, Kirsten Gade Malmos, and Thomas Vosegaard Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05416 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019

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Analytical Chemistry

Analytical evaluation of low-field 31P NMR spectroscopy for lipid analysis Boris Gouilleux*, Nichlas Vous Christensen, Kirsten G. Malmos, and Thomas Vosegaard* Interdisciplinary Nanoscience Center and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, DK8000 Aarhus C, Denmark. ABSTRACT: We investigate the potential of 31P NMR with simple, maintenance-free benchtop spectrometers to probe phospholipids in complex mixtures. 31P NMR-based lipidomics has become an important topic in a wide range of applications in food- and health-sciences, and the continuous improvements of compact, maintenance- and cryogen-free instruments opens news opportunities for NMR routine analyses. A prior milestone is the evaluation of the analytical performance provided by 31P NMR at low magnetic field. To address this, we assess the ability of state-of-the-art benchtop NMR spectrometers to detect, identify and quantify several types of phospholipids in mixtures. Relying on heteronuclear cross-polarization experiments, phospholipids can be detected in 2 h with a limit of detection of 0.5 mM at 1 T and 0.2 mM at 2 T, while the headgroups of PC, PE, PI, PS and PG can be unambiguously assigned based on 2D 1H-31P TOCSY spectra. Furthermore, two quantitative methods to obtain absolute concentrations are proposed and discussed, and the performance is evaluated regarding precision and accuracy.

High-field (HF) NMR is a rapidly emerging analytical tool in lipidomics studies. The high analytical reproducibility and non-destructive character together with the structural, dynamic, and direct quantitative insight provided by NMR warrant the use of this spectroscopy in lipidomics, as recently demonstrated in several applications ranging from disease diagnostic to food science.1–9 However, HF NMR is not generally regarded as a routine analytical tool due to obvious economic and practical reasons. With the recent breakthroughs in the development of low-field (LF) benchtop spectrometers, based on 1-2 T permanent and cryogen-free magnets, this need for a more accessible NMR spectroscopy may be fulfilled.10,11 The last years have witnessed the use of LF NMR spectroscopy in lipidomics through studies in food science12 and of surfactants.13 These promising applications are mainly based on 1H NMR spectra of highly concentrated lipids in different matrices, whose resulting spectra are congested and suffer from peak-overlaps. Usually, the problems associated with this resolution loss are alleviated by using chemometric tools and/or 2D experiments to obtain significant and reliable insights.14–17 While heteronuclear NMR is widely used in lipidomics studies, this type of experiment has not yet been exploited in LF NMR for such applications despite the ability of the modern benchtop spectrometers to probe different heteronuclei. Especially, 31P provides interesting and relevant features such as a high chemical shift dispersion compared to 1H, 100 % of natural abundance, and a high gyromagnetic ratio ensuring high sensitivity even at low field. Hence, 31P NMR has proved to be a valuable probe for the analysis of

phospholipids (PL) in different matrices such as food samples, body fluids, tissue extracts, and cells. 18–22 We present here a detailed 31P NMR study at low magnetic field to investigate the potential of this analytical tool for routine lipidomics applications. The analytical performance of benchtop 31P NMR experiments is evaluated and the ability for the assignment of PL headgroups and the quantification of PL in mixtures is discussed. Two types of experiments have been investigated to fulfill the aforementioned purposes: a standard one-pulse 31P NMR experiment with protondecoupling and 1H-31P heteronuclear cross-polarization (Het-CP) experiments performed in a one-or twodimensional fashion. This paper is outlined as follows: First, the limit of detection for a standard one-pulse 31P NMR experiment with proton-decoupling is determined using samples containing one phospholipid type. Then we highlight the further sensitivity enhancement enabled by the implementation of a 1H-31P heteronuclear crosspolarization (Het-CP). Besides a 2-3 times SNR enhancement typically measured on PL samples, such a Het-CP experiment turns to be stable towards variations in the 3JPH-couplings ranging from 1-10 Hz in the phospholipid P-O-CH2 moiety. The robustness of the CP experiment to off-resonance and miscalibration effects is asserted to ensure the suitability of the pulse-sequences in routine conditions. Furthermore, the Het-CP block can easily be turned into a 2D 1H-31P TOCSY experiment, which correlates the chemical shifts of all the protons involved in the PL headgroup to the phosphorus nucleus.23 The

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resulting traces along the 1H-dimension exhibit unique fingerprints so that several groups of PL can be distinguished. This assignment method is particularly relevant since the 31P chemical shifts change significantly when changes in the experimental parameters or chemical composition occur.24–26 Finally, the analytical performance of 31P benchtop NMR as a quantitative tool is evaluated for the relative and absolute quantification of individual PL types in mixtures. Two approaches delivering absolute concentrations are proposed and discussed and the performance pertained to the quantification is assessed by calculus of precision and accuracy.

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exchange of any paramagnetic ions in the lipid sample thereby avoiding detrimental line-broadening in the resulting spectra. Diaminocyclohexane diaminetetraacetic acid (CDTA) was suspended in MQ water. CsOH·H2O (s) was added stepwise after several additions all CDTA was dissolved, pH was adjusted from initially 6.4 to 10 with CsOH·H2O (s). The volume was adjusted giving a final concentration of 0.2 M CDTA. It is worth mentioning that the use of deuterated chloroform was not required to perform low-field experiments since the benchtop spectrometer has a 19F external lock system. Deuterated chloroform was only necessary for subsequent high-field NMR experiments.

METHODS NMR spectrometers. Low-field NMR spectra were recorded with two benchtop NMR spectrometers (Spinsolve, Magritek, Germany) operating at ca 1 T and 2 T. The magnetic fields lead to B0 frequencies of 43.5 MHz (1H) and 17.6 MHz (31P) for detection at 1 T and 80.5 MHz (1H) and 32.6 MHz (31P) at 2 T. The B0 magnetic fields were produced by a cryogen-free magnet based on a Halbach design compatible with standard 5 mm NMR tubes. An External lock-system operating on the 19F frequency enables the use of non-deuterated solvents without large frequency drifts. The working temperature was fixed at 28.5 °C. The magnet was shimmed through the automatic algorithm provided by Spinsolve software leading to linewidths (full width at half height) in a range of 0.5 – 0.7 Hz measured on a reference H2O/D2O sample. High-field NMR experiments were performed on a Bruker 500 MHz Avance III spectrometer with an actively shielded standard bore 11.7 T magnet. The spectrometer was operated with a 5 mm HX liquid state NMR probe. Phospholipids and chemicals. Throughout the paper the phospholipid groups are referred as follows: phosphatidylcholine (PC); phosphatidyl-ethanolamine (PE); phosphatidylinositol (PI); phosphatidylserine (PS); phosphatidyl-glycerol (PG); lysophosphatidyl-choline (LPC); lysophosphatidyl-ethanolamine (LPE). These PL groups are sketched in Scheme 1. The standards used in this study were 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (POPC), 1,2di-O-tetradecyl-sn-glycero3-[phosphor-rac (1glycerol)] (TDPG), 1,2-dioleoyl-snglycero-3-phospho-L-serine (DOPS). The lipid sample was a certified sample of 20% L-α-lecithin. All lipids were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Two compounds were used as quantitative reference: diphenyl phosphate (DPP) and tributyl phosphate (TBP) (Sigma Aldrich), both provided a purity of 99 %. Sample preparation. Phospholipid samples were prepared with a ternary solvent system: CDCl3/MeOH/cesium cyclohexane diamine tetraacetic acid (CsCDTA) (aq) with the respective volumes: 714/285/75 μL. The use of such ternary solvent systems is a determining feature for the resolution of the NMR experiments.27,28 The CsCDTA complex enables the

Scheme 1. Schemes of phospholipid groups and reference compounds involved in this study. Lyso-phospholipids such as LPC and LPE refer to molecules where R1 or R2 are H atoms. NMR experiments and parameters. NMR pulse sequences used throughout this study are sketched in Figure 1. Proton-decoupled 31P NMR (31P{1H}) experiments (Figure 1a) were performed with the following parameters: the 90° flip angle was achieved with pulse duration of 27.5 and 33.5 μs with the 1 and 2 T spectrometer, respectively. The resulting 31P FID was recorded with 4 K points separated by a dwell-time of 200 μs leading to an acquisition time of 0.82 s. Proton composite-pulsedecoupling (CPD) was applied during the detection. Decoupling is only applied during acquisition (inverse gated decoupling) to avoid distortions of the intensities due to the nuclear Overhauser effect.29 The heteronuclear transfer of spin order was achieved by cross-polarization where the Hartmann-Hahn condition is achieved when the following is fulfilled


1 |𝛾 𝐵 ― 𝛾𝑆 𝐵1𝑆| < 𝑓𝑖𝑛𝑡. 2𝜋 𝐼 1𝐼

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Analytical Chemistry

Figure 1. Pulse sequences used in the study. (a) 31P one-pulse experiment with proton CPD (31P{1H}). (b) 1H-31P heteronuclear crosspolarization experiment (Het-CP) with 31P detection under proton CPD. (c) 2D 1H-31P heteronuclear TOCSY experiment with 31P detection under proton CPD. Thin filled rectangles represent hard 90° pulses while thick open pulses correspond to 180° refocusing pulses. The spinlock is achieved with a WALTZ-16 block applied on both channels, flanked by a pair of trim-pulses. In all the experiments, CPD is only applied during the acquisition (inverse gated decoupling) to avoid NOE effect, which would disturb quantitative results.

Here, 𝛾𝐼𝐵1𝐼/2𝜋 and 𝛾𝑆𝐵1𝑆/2𝜋 correspond to the RF fields applied on each channel while 𝑓𝑖𝑛𝑡 is the spin-spin interaction frequency, e.g. dipolar and scalar couplings in solid and liquid state respectively. While the HartmannHahn condition is met with a relative ease for solid-state experiments because of the large dipole-dipole interactions leading to values of 𝑓𝑖𝑛𝑡 in the kHz range, the situation is more difficult in liquids due to the typical low values of the scalar coupling. In the context of phospholipids, 3JPH-couplings are within a range of 1 – 10 Hz, which makes the experiment far from straightforward. To address this issue, the 1H-31P heteronuclear crosspolarization was yielded in a robust manner with a WALTZ-16 sequence following 1H excitation (Figure. 1b). The effect of such multi-pulse sequences is that the effective nutation frequency is constant over a large range of parameters, implying that the Hartmann-Hahn condition is met for a larger range of RF field mismatch and chemical shift offsets than for a standard continuous-wave spinlock. As a result, these sequences have already proved their suitability for 1H-13C and 1H-31P transfers in liquidstate experiments.23,30,31 Following a 90°y 1H-excitation achieved with 11.9 and 13 μs as pulse durations at 1 and 2 Tthe super-cycled WALTZ-16 sequence employs a pulse duration of 57.5 μs for the nominal 90° flip angle, delivering an RF field along the X-axis of 4.3 kHz on both channels. The resulting 31P FID was recorded in similar manner than for 31P{1H} experiments. To correlate 31P nuclei to the 1H-networks involved in the headgroups of phospholipids, the Het-CP block was used as the mixing step of a 2D 1H-31P TOCSY experiment as sketched in Figure 1c. In this pulse-sequence, 1H chemical shift is encoded during the t1-evolution period while JPH coupling is refocused. 31P chemical shift was then monitored during t2-detection under proton decoupling. Besides the inherent low sensitivity of benchtop NMR, the short T2-relaxation of PL occurring during the evolution period implies the need to tune experimental parameters to maximize the sensitivity. Hence, the signal in F2 was acquired in shorter duration: 2 K points with a dwell time of 200 μs leading to an acquisition time of 0.41 s. The indirect dimension was sampled with a t1max lower than

the T2 pertained to PL, but long enough to enable PL group distinction. The t1max – whose values are given in figure captions - are obtained by recording the indirect FID with a short dwell-time leading to large spectral widths in ppm along F1, e.g. 20.7 ppm at 1 T. Such a spectral width is larger than necessary, a situation commonly encountered in LF NMR. However, this situation could be turned as an advantage to yield 2D data in a phase-sensitive mode without the need of any STATES or TPPI procedures.32 Indeed, the double absorption line-shape can be easily obtained by recording the 2D interferogram with an amplitude modulation in t1 whereas the frequency discrimination is obtained by translating the receiver frequency in F1 at the edge of the spectrum. The imaginary part of the (t1, F2) map was then discarded prior to the second Fourier transformation to yield the desired double absorption line shape. This procedure circumvents the acquisition of a whole hypercomplex interferogram leading to a significant time-saving. Although this method is not usually recommended in HF NMR due to potential offresonance effects, such issues become not relevant at low magnetic field. Moreover, the robustness of the WALTZ block against offset effects – presented later in Figure. 2 ensures the suitability of this procedure. To get rid of undesired coherences and ease the 2D-phasing, trimpulses of 1 ms were applied on both sides of the WALTZ-16 block. Gaussian apodization (1.3 Hz in F2 and 2.6 Hz in F1) and a four-times zero-filling were applied in both dimensions prior to the double Fourier transformation. The matrix size after processing was (8192 X 512) (F2 X F1). 1H and 31P chemical shifts were referenced to TBP whose CH3 signal resonates at δ(1H) = 0.90 ppm (relative to TMS) in F2 and at δ(31P) = -1.73 ppm (relative to 85 % H3PO4) in F1. NMR processing and deconvolution. All NMR data were processed in Mnova (v.12.0.2, Mestrelab Research), except the 2D data, which were treated by a home-written Prospa macro within Spinsolve Expert (Magritek, Germany) with parameters described above. 1D spectra were zero-filled to 65536 points, manually phased, and baseline corrected using the Whittaker smoother algorithm provided by Mnova. A 0.6 Hz Gaussian



apodization was applied if not otherwise mentioned. Peakpicking was performed manually and deconvoluted using the Mnova line fitting tool. The line-fitting algorithm optimized the following parameters: peak-height, linewidth and the Lorentzian/Gaussian ratio. The latter parameter took into account the Gaussian character of the signal arising from field inhomogeneities and Gaussian apodization.

36

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126

238

298

Mixing time was 90 ms for Het-CP. “nd” stands for nondetected.

RESULTS AND DISCUSSION Limit of detection and sensitivity enhancement. The sensitivity of the standard 31P{1H} experiment is evaluated on a series of samples containing DMPC in a concentration range from 0.5 to 36 mM. Table 1 shows the signal-to-noise ratio (SNR) measured in a 2 h experiment (6000 scans) for each concentration. Since the application of apodization functions involves a trade-off between sensitivity and resolution, SNR is reported for spectra processed without (no LB) and with an exponential apodization of 1 Hz (LB), which approximately corresponds to matched apodization.33,34 It should be noted that an improved resolution can be obtained by using a bell-shaped window function.33 However, such treatment render the intensities not fully reliable and is therefore not applied. When considering the limit of detection (LOD) as the minimal concentration to yield SNR = 3,35 a 2 h 31P{1H} experiment provides a LOD of 1.5 mM. To further improve the sensitivity of 31P benchtop NMR, methods based on 1H-31P magnetization transfer are considered. Although methods based on INEPT blocks are the most popular and widely used in liquid-state NMR, a heteronuclear cross-polarization approach is used in this study for several reasons. First, Het-CP provides a higher sensitivity enhancement compared to INEPT since all the protons within the headgroup of PL are involved in the polarization transfer. Second, INEPT requires a careful matching of the spin-echo delay to the J coupling. This is problematic due to the range of 3JPH couplings (1 – 10 Hz) observed in the P-O-CH2 moieties. This makes the use of INEPT inappropriate for detection of different phospholipids in mixtures, while Het-CP achieved a fairly homogenous polarization transfer regarding the Jcouplings variations for mixing times from 70 to 180 ms (Figure S1). Table 1. Signal to noise ratios from 31P {1H} and Het-CP. 31P{1H}

Het-CP

Concentration (mM)

no LB

LB=1Hz

no LB

LB=1Hz

0.5

nd

nd

nd

4

1.5

3

4

6

11

3

5

8

13

18

10

14

20

40

51

25

49

72

148

171

Figure 2. (a) Off-resonance effect evaluated by measuring the signal integration as function of the offset frequency on the 31P channel. (b) Impact of the pulse-miscalibration evaluated by measuring the signal integration while the pulse amplitude of the 31P channel is modified. Het-CP experiments were performed with a spinlock mixing time of 90 ms on a sample of tri-isobutyl phosphate dissolved in CDCl3. The signal integrations were normalized to the on-resonance experiment without miscalibrations.

Applying the Het-CP experiment on the DMPC samples mentioned above leads on average to a 2.6 times SNR enhancement compared to the one pulse experiment and results in a LOD of 0.5 mM in 2 h. It should be noted that in this sample 31P and 1H T1 relaxation are not so different: 4.0 and 2.9 s, respectively. Hence, the sensitivity enhancement mainly arises from the CP transfer. The LOD determined at 1 T can be extrapolated to 0.2 mM for a 2 T magnet when considering SNR ∝ B03/2, which is a reasonable approximation for magnets of similar designs (vide infra). Thus, the sensitivity of 31P benchtop NMR is suitable for lipidomics applications involving lipid concentrations in the order of mM. It should be noted that due to the nature of NMR, we may improve the SNR by the factor √t, where t is the experiment time. Thus, in order to improve the LOD by a factor 2 (e.g. from 0.2 mM to 0.1 mM) implies recording four times as many scans corresponding to 8 h experiment time. The robustness towards offset effects and pulse miscalibrations of the Het-CP experiment is addressed from two series of Het-CP experiments. (i) Increasing the offset frequency between the signal of interest and the carrier frequency and (ii) by varying the pulse amplitude of one channel (here 31P) to mimic different degrees of miscalibrations. Figure 2a shows that the off-resonance


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Analytical Chemistry effect is not significant until 1500 Hz, i.e. 85 ppm at 1 T, which is well outside the PL chemical shift range (-20 to +10 ppm). Hence, in the context of PL mixtures, the polarization transfer is not chemical shift dependent and no specific care is necessary to fix the carrier frequency. Furthermore, Figure 2b reveals a good robustness of the WALTZ-16 spinlock sequence to RF miscalibrations. These properties allow to record sample series without the need for elaborate pulse-calibrations prior to each run, an important asset for the use of Het-CP as a routine tool.

TBP is used to reference 1H and 31P chemical shifts. 2D spectra are recorded in a phase-sensitive mode to further improve the resolution in both dimensions (see Methods section). Traces along F2 deliver resolution to distinguish each signal without peak-overlaps (Figure 3) while the traces in F1 exhibit unique profiles, which enable the discrimination of the present PL groups. Moreover, inspection of F1-traces (not shown here, but see Figure 4e for a later discussion), shows the low level of t1-noise pointing out the high temporal stability of the hardware.

Identification of lipids and relative composition of phospholipid mixtures. Identification of different phospholipids from the NMR spectra is an essential part of an NMR-based lipidomics procedure. Relying on a linewidth of typically 1.2 Hz, i.e. 0.07 and 0.035 ppm at 1 T and 2 T, respectively, benchtop spectrometers warrant sufficient resolution to distinguish several phospholipid groups with standard 1D experiments. However, this is not always sufficient for an unambiguous identification. 31P chemical shifts are highly sensitive to pH, temperature and other parameters related to the sample preparation. Although partly removed by the present solvent system, such fluctuations are inevitable in non-standard samples impeding the use of 1D 31P spectra as a reliable tool to assign PL groups.

Following this first example, the same method is applied on a sample of 60 mg α-lecithin dissolved in the same ternary solvent system. The lipid sample contains five PLs with the following %mole: PC (35.21 %), PI (17,32 %), PE (32.87 %), LPC (1.62 %), LPE (1.98 %) and PA (8.84 %). The few remaining percent to reach 100% correspond to inorganic and unknown phosphorus containing compounds. Figure 4a shows the resulting 2D spectrum recorded at 1 T in 12 h. It is worth combining the 2D data obtained on the model and lecithin samples to review all the PL 1H-fingerprints encountered in this study (Figure 4e). This points out a clear distinction between the PL groups based on the variations in proton chemical shifts: PC (δ(1H): 3.6, 4.0, 4.2 and 5.2 ppm), PG (δ(1H): 3.6, 3.8 ppm), PS (δ(1H): 3.9, 4.2 and 5.2 ppm) and PE (δ(1H): 3.1, 3.9 and 5.2 ppm) as well as the relative intensities of the lines in each spectrum. PI also seems to have a unique signature, nevertheless a more sensitive experiment would be beneficial to confirm this profile. A 2D TOCSY experiment performed at 2 T provides sufficient sensitivity enhancement to confirm the F1-pattern for the PI group (Figure 4b). Moreover, the increased sensitivity at 2 T enables a significant reduction of the experiment duration (from 12 to 6 h) with a similar line width in ppm with the respect of F1.

Figure 3. 2D 1H-31P TOCSY spectrum recorded on a mixture of standard phospholipids with approximate concentrations of 25 mM. The spectrum was acquired in 12 h: 100 t1-increments with 432 scans and a repetition time of 1 s. The spectrum is plotted in phase-sensitive mode. The top trace corresponds to the F2-trace highlighted by the dashed line on the spectrum.

An appealing solution is to probe the correlation between 31P nuclei with protons involved in the glycerol and headgroup parts, where the latter are characteristic for each PL group. As the 1H chemical shift is robust towards the above-mentioned experimental issues, such 31P-1H correlations enable an unambiguous assignment of PL in mixtures.24–26 In this context, the previously described HetCP block is turned into a 2D 1H-31P TOCSY experiment. Figure 3 shows an example of such 2D spectrum recorded in 12 h at 1 T on a model sample including DMPC, DOPS and TDPG lipids at concentrations of 25 mM for each lipid.

Following the assignment, 31P{1H} experiments are performed on the same lipid sample to yield quantitative information about the PL composition. A repetition delay of 2 s ensures the full longitudinal relaxation of all PL signals. The TBP signal has a 31P relaxation time of T1=14 s at 1 T and will be partly saturated. However, this is not important at this point, as it is here only used for referencing the ppm scale. 1 and 2 T experiments provide a sufficient sensitivity and resolution to probe the main compounds: PC (δ31P: -1.38 ppm), PE (δ31P: 0.60 ppm) and PI (δ31P: -0.96 ppm) while the minor component LPE and LPC are only detected at 2 T, nonetheless, with a high degree of overlaps (Figure 4c and 4d). A 3.2 times SNR enhancement is observed for the experiments carried out at 2 T compared to 1 T, leading to 90 (PC), 35 (PI) and 48 (PE) as SNR at 2 T in 2 h. As a comparison, a 23 min experiment recorded at 11.8 T delivered 525 (PC), 97 (PI) and 346 (PE) as SNR. A comparison of low and high-field spectra is displayed in Figure S2. After signal deconvolution, the relative composition in %mole is given in Table 2. These 2 h experiments lead to a precision better than 8% at 1 T and 3 % at 2 T. The improvement of the precision at 2 T is consistent with the higher SNR measured at this field.



Furthermore, the accuracy is better than 1 % on both spectrometers for the PC signal, which is almost well resolved. Unfortunately, the accuracy significantly decreases within the more congested spectral regions, e.g.

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PE, for which 1 T data leads to a high relative error of 19 %. The resolution enhancement provided at 2 T alleviates this issue so that a quantification of the PE group is achieved with an accuracy of 1.7 %.

Figure 4. 2D 1H-31P TOCSY spectra of a sample of 60 mg of α-Lecithin, recorded at 1 T in 12 h (a) and at 2 T in 6 h (b). Both experiments provided approximately the same line-width (in ppm) with the respect of the indirect dimension, t1max = 111 and 64 ms at 1 and 2 T, respectively. 2D data were acquired with a mixing time of 90 ms, repetition time of 1 s and plotted in phasesensitive mode. (c) 31P{1H} spectrum of the same sample recorded in 2 h at 1 T and (d) at 2 T. The repetition time of 1D experiments was fixed to warrant a full longitudinal relaxation of PLs. (e) Molecular structures of relevant PL groups and their corresponding 1H-traces extracted from the 2D 1H-31P TOCSY spectra recorded at 1 T.

Table 2. Phospholipid content in %mole of an α-lecithin sample obtained from 2 h 31P {1H} at 17.6 and 32. 6 MHz. 1 T experiment

2 T experiment

PL groups

%mole

Precision (RSD)

Accuracy

%mole

Precision (RSD)

Accuracy

PC

34.7%

1.7%

1.4%

35.0%

1.6%

0.5%

PI

17.8%

7.9%

2.5%

18.7%

2.7%

8.0%

PE

39.2%

2.7%

19.1%

33.4%

2.3%

1.7%

LPC

nr

-

-

na

-

-

LPE

nr

-

-

na

-

-

PA

nr

-

-

na

-

-

A repetition time of 2 s was sufficient to warrant full relaxation of PLs. Integrations were performed by deconvolution as explained in the experimental section. Precision was calculated as residual standard deviation (RSD) with three successive experiments while accuracy corresponds to the trueness based on a quantitative experiment carried out at 500 MHz. “nr” stands for “non-resolved” and “na” when the signal can be peak-picked, but without unambiguous assignment.

Absolute quantification of phospholipids. Besides measuring the relative PL composition, it will in some cases be necessary to measure absolute PL concentrations, especially for samples where no prior knowledge exists. Working on an absolute scale requires the use of an internal standard in a known concentration which undergoes full relaxation within the repetition time for quantitative measurement. Several reference compounds

commonly encountered in 31P NMR have been investigated and two of them (DPP and TBP) provide chemical shifts in empty regions of the 31P NMR spectra of lipids thereby avoiding overlaps with signals of interest. However, DPP and TBP both provide long 31P T1 relaxation times at 1 T: 4.1 and 14 s, respectively, which is not optimal considering the short 31P T1 values for PLs, i.e., 0.37 s. This feature hampers the necessary full longitudinal relaxation between two


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Analytical Chemistry scans within a reasonable repetition time, yet we have not managed to find suitable alternatives with short 31P T1 values. To circumvent this problem, two alternative methods are here proposed and evaluated. The applicability of these approaches is evaluated on a lipid sample consisting of 30 mg α-lecithin and 10 mg of TDPG. The performance of the quantification is evaluated on two targeted PL groups: PG and PC. This allows to assess accuracy and precision on a well-resolved peak (PG) as well as on a slightly overlapped signal (PC). The first quantitative approach relies on 31P {1H} experiment in combination with a relaxing agent to increase the spin-lattice relaxation rate and hence a decreased T1. Gadoteric acid (DOTAREM, Guerbet, Roissy, France) is used for this purpose. The tradeoff with this approach is a concomitant shortening of the spin-spin T2relaxation causing line broadening. To investigate this in detail, phosphorus T1 values and linewidths have been measured with several DOTAREM concentrations to determine the concentration leading to a sufficient T1 decrease while maintaining an acceptable linewidth. By plotting the measured T1 values as a function of the relaxing agent concentration, two different linear trends are successively observed (Figure S3). This result is consistent with previous reported observations.36 It has turned out that a concentration ratio of 1:100 (DOTAREM/DPP) is the ideal compromise. For the samples studied here, containing 15 mM DPP as internal reference, this implies a concentration of 0.15 mM DOTAREM. The 31P T1 value at 1 T of DPP is reduced to 1.4 s in the lipid mixture so that subsequent quantitative experiments are carried out with a repetition time of 7.5 s resulting in an experiment time of 8 h for 3840 scans. The second quantitative approach relies on the Het-CP experiment (Figure 1b). In this experiment, 1H spins are excited and magnetization transferred to 31P. Therefore, the repetition time can be adjusted according to the proton longitudinal relaxation. In addition, the increased SNR provided by Het-CP (Table 1) should improve the repeatability of the subsequent quantification. A central feature with this approach is that the magnetization transfer efficiency from 1H to 31P depends on the PL type. As such, Het-CP cannot be applied directly to deliver quantitative data. However, by using external calibration curves for each PL of interest the difference in transfer efficiencies can be accounted for. This approach, already reported in quantitative 2D NMR provides a typical accuracy and precision of about 5 %.37,38 An External calibration curve is established from a single series of four calibration samples containing two PL standards (PG and PC) with concentrations ranging from 4.7 to 17.5 mM, 4 μL of TBP (12 mM) is added as internal reference, which is here preferred to DPP since the latter does not provide a 3JPH 1H-31P coupling required for cross-polarization. Quantitative experiments obtained in 8 h employing 4000 scans and a repetition time of 7.2 s (full 1H-relaxation of TBP), are performed for each concentration. The resulting curves (Figure S4) demonstrate the high linearity with R2 > 0.99. The PG standard TDPG, present in the lipid sample

may be used for calibration. That would be more problematic for PC since the signal arising from PC lipids is congested with signals from mixed fatty acids present in lecithin. POPC is the most representative available standard for calibration as this PC lipid provides a molecular weight: 760 g/mol close to the average value of PC in lecithin: 770 g/mol. The analytical performance of the quantification is evaluated through the following criteria: (i) Quality of the resulting data in terms of sensitivity and resolution; (ii) precision and (iii) accuracy.

Figure 5. Quantitative spectra recorded with proton 31P{1H} experiment in presence of relaxing agent (a) and with the HetCP experiment (b). Both spectra were recorded in 8 h.

In the following, accuracy is calculated as the relative error between the concentration obtained by the evaluated method and the concentration measured with a quantitative 31P experiment performed at 500 MHz while the precision is assessed by calculating the relative standard deviation on five successive experiments. The comparison of Figures 5a and 5b highlights the improved resolution of the Het-CP spectra compared to the singlepulse method where line-broadening is caused by the relaxing agent DOTAREM. Thus, considering criterion (i), the use of Het-CP delivers data with a significant higher quality and thus warrants a lower limit of quantification. Regarding the precision (criterion (ii) and Table 3), both methods provide RSD lower than 5 % for the quantification of PG and PC. Furthermore, Het-CP delivers more precise (RSD ≤ 3 %) results compared to the one-pulse 31P (RSD ≤ 5 %). This result is well explained by the sensitivity enhancement provided by Het-CP since the repeatability is inversely proportional to the SNR. Moreover, the linefitting performed on the resulting one-pulse spectra is complicated by the DOTAREM-induced line-broadening leading to a higher residual error over the integration pattern. In regard to criterion (iii) absolute concentrations measured on PG show a slightly better accuracy of Het-CP (2 %) compared to the one-pulse experiment (4 %). This notable good accuracy could be explained by the fact that external calibration curves are here plotted with the relative integration between phospholipid and reference signals instead of the absolute integration. This procedure limits the impact of matrix effects between model and real



samples and in addition circumvents temporal spectrometer instabilities. However, the accuracy of PC quantification points out limitations of quantitative HetCP. The issue being that the PC resonance arises from several different lipids with a PC headgroup but with slightly different NMR properties, therefore a standard curve recorded on pure POPC does not recapture all transfer efficiencies for PC lipids in the sample of interest. Rather, the observed low accuracy of only 9 % is not surprising. On the contrary, the 31P{1H} applied, together with relaxing agents, is more versatile in such a case and delivered quantitative result with accuracy better than 5 %. In summary, quantitative Het-CP delivers a better analytical performance when standards are available for calibration while a one-pulse experiment is more versatile and accurate otherwise. Table 3. Absolute concentrations for TDPG and PC measured with the two described quantitative methods. 31P {1H}

1H-31P

Het-CP

Compounds

PG

PC

PG

PC

SNR

31

17

70

43

Mean concentration (mM)

14.1

8.0

13.3

7.3

Precision (RSD)

3.38%

4.95%

2.19%

2.80%

Accuracy

3.99%

0.50%

1.94%

8.67%

CONCLUSION The analytical performance of low-field 31P NMR spectroscopy for lipidomics analysis has been evaluated and discussed. First, the LOD obtained by 2 h of 1H-31P cross-polarization is 0.5 mM at 1 T and can be extrapolated to 0.2 mM at 2 T. The Het-CP block can also be used as a 2D TOCSY experiment to unambiguously assign the major PLs in a robust manner not affected by experimental parameter and sample preparation changes. This also enables the relative quantification of PL groups in a lipid sample with an average precision and accuracy better than 5 % and 8 %, respectively at 1 T, and better than 3 % and 4 % with a 2 T spectrometer. In addition to the relative composition, the absolute concentration of a particular PL component can be obtained with a precision ranging from 5 to 3 % and with accuracy better than 2 to 4 % within reasonable experiment time, depending on the methodology. This overall performance makes 31P NMR at low-magnetic field a promising routine tool for NMRbased lipidomics applications in food (and biomedical) sciences. Furthermore, there is still room for improvement, especially for the assignment of minor PL groups in mixtures and to speed up the 2D experiments. In this context, approaches such as non-uniform sampling would significantly shorten the experiment duration or improve the current sensitivity by averaging more scans per t1increment.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. CP transfer versus mixing time; Comparable high and lowfield 31P spectra; T1 values of internal reference versus concentration of relaxing agents; external calibration curves (PDF)

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (B.G.), [email protected] (T.V.)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial support from the Danish Minstry of Higher Education and Science (AU-2010-612-181), the European Commission (H2020 Future and Emerging Technologies grant 731475 and Research Infrastructures grant 731005), Novo Nordisk Fonden (NNF16OC0021110), and Carlsbergfondet (CF15-211) is acknowledged. We are grateful to Magritek GmbH and Dr. F. Cassanova for providing access to the Magritek 80 MHz spectrometer.

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Analytical Evaluation of Low-Field 31P NMR Spectroscopy for Lipid

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