Quantitative Profiling of Endogenous Fat-Soluble Vitamins and

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Quantitative profiling of endogenous fat-soluble vitamins and carotenoids in human plasma using an improved UHPSFC-ESI-MS interface Filomena Petruzziello, Alexandre Grand-GuillaumePerrenoud, Anita Thorimbert, Michael Fogwill, and Serge Rezzi Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 04 Jun 2017 Downloaded from http://pubs.acs.org on June 5, 2017

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Analytical Chemistry

Quantitative profiling of endogenous fat-soluble vitamins and carotenoids in human plasma using an improved UHPSFCESI-MS interface Filomena Petruzziello*,‡,†, Alexandre Grand-Guillaume Perrenoud‡,†, Anita Thorimbert†, Michael Fogwill§ and Serge Rezzi† †

Nestlé Institute of Health Sciences, Molecular Nutrition Group, EPFL Innovation Park, H, 1015 Lausanne, Switzerland. Waters Corporation, Core Research. 34 Maple St. Milford, MA, 01757, USA. ‡ These authors contributed equally. *[email protected] §

ABSTRACT: Analytical solutions enabling the quantification of circulating levels of liposoluble micronutrients such as vitamins and carotenoids are currently limited to either single or a reduced panel of analytes. The requirement to use multiple approaches hampers the investigation of the biological variability on a large number of samples in a time and cost efficient manner. With the goal to develop high-throughput and robust quantitative methods for the profiling of micronutrients in human plasma, we introduce a novel, validated workflow for the determination of 14 fat-soluble vitamins and carotenoids in a single run. Automated supported liquid extraction was optimized and implemented to simultaneously parallelize 48 samples in 1 hour and the analytes were measured using ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry in less than 8 minutes. An improved mass spectrometry interface was developed to control the density of the chromatographic effluent on its route towards and into the ion source. The optimized interface resulted in improved spray plume stability and matrix compounds solubility leading to enhanced robustness of the transfer capillary. An interface hardware minimizing the post-decompression volume and a specific make-up solvent conditions were both developed to provide suitable analytical repeatability and improved the detection sensitivity. The developed methodology gives recoveries within 85-115 %, as well as within and between-day coefficient of variation of 2 and 14 %, respectively.

Fat-soluble vitamins (FSV) and carotenoids are micronutrients naturally occurring in food and are required by the human body to maintain a broad range of physiological and biochemical functions1, 2. Both FSV and carotenoids need to be obtained from diet on a regular basis in order to fulfill healthy nutritional and metabolic requirements. Their analysis in biological samples such as blood provides important information to assess the nutritional status of an individual2 and thus to identify potential deficiencies that have been associated to a broad range of pathophysiological conditions such as osteoma-

lacia (vitamin D)3, 4, night blindness (vitamin A)5, 6, increased cellular oxidative stress (vitamin E)7-9 and hemorrhage (vitamin K)10, 11. However, the occurrence of numerous structural or functional homologs within each family of FSV (vitamers) and carotenoids (isomers) as well as their respective concentration ranges make difficult their simultaneous analysis in blood samples12, 13. These constraints have led to the development of different analytical protocols able to target either single or a restricted panel of FSV and carotenoids14-17. Therefore, the absence of a fast single assay for the profiling of such

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compounds in biofluids hampers the systematic acquisition of quantitative information on FSV and carotenoids in nutritional epidemiology research with respect to throughput limitations and sample volume requirement for multiple analytical protocols. Irrespective of the different vitamers sub-classes, sample preparation available methods require convergent procedures involving an initial protein precipitation step mostly followed by liquid-liquid extraction (LLE)16-20 sometimes using hazardous solvents including n-hexane18, 20, chloroform17, or dichloromethane19, 20. The LLE step is difficult to automate and parallelize21 without complex robotics20 and is mostly performed manually in series. Alternatives such as solid phase extraction (SPE) or derivatization are more scarcely reported and are generally used as part of the LLE protocols15. Surprisingly, supported liquid extraction (SLE) that has been already applied to fat-soluble vitamins in plant or food materials12 hasn’t been reported for FSV and carotenoids extraction from biological matrices. SLE uses high-surface-area and chemically inert diatomaceous earth as a stationary vehicle for the aqueous phase of the LLE experiment. The aqueous phase very easily adsorbs onto the surface to form a retained and widely dispersed film containing the analytes of interest. The analytes are then rapidly extracted using an immiscible organic solvent that percolates within the support. The SLE procedure provides rapid extraction with equivalent or even better recoveries related to LLE. Additionally, solutions exist for SLE automation and parallelization of sample preparation using dedicated pre-packed multi-well plate supports. Downstream to sample preparation, analytical procedures for FSV and carotenoids show more diversity. Separation of vitamers is mostly performed using liquid chromatography (LC) operated in either normal phase (NPLC), reversed phase (RPLC), or non-aqueous reversed phase (NARP) modes whereas detection is ensured using hyphenation to UV, fluorescence, or mass spectrometry (MS) detection12, 13, 23-26. The use of non-polar columns packed with sub-2 µm fully porous particles 15, 19 or sub-3 µm superficially porous particles16, 17 and a highly organic mobile phase have improved the analytical throughput of FSV analysis by ultra-high performance LC (UHPLC). Yet, these methods remain limited to a few compounds or struggle to achieve suitable selectivity and sensitivity for proper quantitation in complex biological matrices. The development of new generation of supercritical fluid chromatography (SFC) instrumentation opens new perspectives for the rapid profiling of broad range of molecules including highly liposoluble species27, 28. Already implemented decades ago for investigating vitamins29, but strongly lacking robustness at that time, SFC exhibits the intrinsic properties required for the analysis of fat-soluble vitamins. With a polarity comparable to those of n-hexane or heptane, SFC mobile phase consists of pressurized carbon dioxide (CO2) blended with a small amount of organic modifier that is able to effectively solubilize species with a wider range of polarities. Furthermore, the SFC mobile phase possesses the solvating power and density of a liquid along with the diffusivity and low viscosity of a gas that allow for fast and kinetically efficient analyte transportation with a reduced column pressure drop30. When combined with the use of stationary phases packed with sub-2 µm particles31-33, ultra-high performance SFC (UHPSFC) achieves performances comparable UHPLC27, 34. Moreover, virtually all stationary phase chemistries, from C18 to pure silica can be used in SFC without any adaptation of the mobile phase composition28, 35, 36. Consequently, SFC shows wide possibilities

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for selectivity tuning which is key when dealing with structurally similar compounds37. Hyphenation with MS provides a supplementary dimension of selectivity and enhanced sensitivity. However, due to the highly compressible nature of the SFC mobile phase, interfacing SFC to atmospheric pressure MS ionization sources requires special care and a built-for-purpose interface38 which must properly manage the CO2 decompression while preventing potential analyte or matrix precipitation along the path towards ionization. For some FSV sub-classes, promising results were very recently obtained using UHPSFC-MS for the semi-quantitative determination of plasmatic concentration of some of vitamin D derivatives and metabolites39 and for the fully validated quantification of E sub-class vitamers in human serum40. In the present contribution, we describe the development of a fully validated workflow allowing for the quantitative determination of human plasmatic concentrations of 14 fat-soluble vitamers belonging to the A, D, E and K classes and carotenoids. Low sample consumption (i.e. 200 µL) and improved throughput were achieved thanks to the integration and optimization of a robotized SLE-based sample preparation with a fast UHPSFC-MS/MS analytical method. This work also details particular care taken to make UHPSFC-MS/MS repeatability and robustness compliant with validation criteria. The validated method required stabilization of chromatographic parameters and implementation of a new and enhanced UHPSFC-MS interface hardware designed to prevent fluid decompression inconsistencies while simultaneously improving sensitivity and lowering solvent consumption. This validated method is well suited for both research and routine deployment of quantitative profiling of FSV and carotenoids in human plasma.

EXPERIMENTAL SECTION Materials, chemicals and chromatographic columns. Analytical or ultra-pure grade reagents were used for these analyses. Isopropanol (IpOH), heptane, ethanol (EtOH), and methanol (MeOH) were provided by Biosolve (ChemieBrunschwig, Basel, Switzerland) while ammonium formate (AmF) and butylated hydroxytoluene (BHT) were purchased from Sigma-Aldrich (Buchs, Switzerland). αtocopherol, α-tocotrienol, β-tocopherol, γ-tocopherol, δtocopherol, δ-tocotrienol, vitamin K1, lutein, zeaxanthin, and β-cryptoxanthin standards were purchased from SigmaAldrich (Buchs, Switzerland) while γ-tocotrienol was purchased from Acros organics (Geel, Belgium). Retinal was purchased from Toronto Research Chemicals (Toronto, ON, Canada) and 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). Isotopically labeled 2H6-25-Hydroxyvitamin D3, 2H7vitamin K, and 2H6-α-tocopherol were purchased from SigmaAldrich (Diegem, Belgium). Tomatidine was purchased from Extrasynthese (Lyon, France). AmF was provided by SigmaAldrich (Buchs, Switzerland). Pressurized CO2 (purity 99.9995%) was purchased from Air Liquide (Düsseldorf, Germany). The extraction was performed using ISOLUTE SLE+ supported liquid extraction 48 well-plate purchased from Biotage (Uppsala, Sweden). Internal standards (IS). 2H6-25-Hydroxyvitamin D3, 2H4vitamin K, and 2H9-α-tocopherol deuterated compounds and exogenous tomatidine were used as quantification IS. Toma-


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tidine was used as IS to quantify β-cryptoxanthin, lutein, and zeaxanthin due to the lack of commercially available related labeled standard. Each stock solution of IS was prepared monthly in EtOH containing 350 mg/L BHT at concentration of 1 mg/mL and stored at -80 °C. A final IS solution was prepared weekly from the stock solutions to reach a final concentration of 10 µg/mL of 2H9-α-tocopherol, 10 µg/mL of tomatidine, 1 µg/mL of 2H4-vitamin K, and 50 µg/mL 2H6-25Hydroxyvitamin D3 in EtOH-BHT and was stored at -80 °C. Vitamin standard stock solutions. A solution of each vitamin standard was prepared monthly at concentration of 1 mg/mL in EtOH containing 350 mg/L BHT and stored at -80 °C. Further dilutions of each stock solution were prepared daily in EtOH-BHT at 10 different calibration levels according to the validation protocol. Human samples. Pooled human plasma with EDTA anticoagulant was purchased from Sigma-Aldrich (Buchs, Switzerland), aliquoted (200 µL), and stored at -80 °C. Sample treatment and supported-liquid extraction (SLE). Oxidative degradation was minimized by using an ethanolic solution of BHT (350 mg/L). Before starting the extraction, 30 µL of internal standard mix was added to 200 µL of plasma. To precipitate the proteins and release vitamins, 400 µL of IpOH was added to the samples. The samples were vortexed and then further diluted with 200 µL of water before being loaded onto SLE 48 well-plates. Automated extraction was performed using an Extrahera robot (Biotage, Uppsala, Sweden) which followed a three-step optimized procedure. First, the sample was adsorbed on the diatomaceous earth plates applying a 5 seconds pulse of 0.2 bar positive pressure followed by a resting time of 5 minutes. Next, the sample elution was performed using two 1000 µL volumes of IpOH/heptane (3/7, v/v) followed by three 1000 µL volumes of heptane. After each elution, the robot applied 0.5 bar of positive pressure on the plate. Finally, the 5 organic elution volumes were combined and dried using a centrifugal evaporator (CentriVap, VWR, Dietikon, Switzerland) for 1 h and reconstituted in 200 µL of IpOH/heptane (3/7,v/v) prior to UHPSFC-MS/MS analysis. UHPSFC-MS/MS instrumentation. The separations were performed using an ACQUITY UPC2 system (Waters, Milford, MA, USA), which consisted of a binary pump, a temperature-controlled autosampler set to 6 °C. The system was equipped with a 10 µL sample loop, a column oven set at 40 °C, and a backpressure regulator (BPR) set at 172 bar. The partial loop with needle overfill mode was used to inject 2 µL of sample. Heptane and IpOH/heptane (1/9, v/v) were selected as strong and weak wash solvent, respectively. The UHPSFC system was hyphenated with a Waters Xevo TQ-S triple quadrupole mass spectrometer. Electrospray ionization (ESI) was employed and operated in positive mode. Nitrogen was used as the desolvation, nebulization, and cone gas while argon was employed as the collision gas. A split-flow interface using a make-up fluid was employed to hyphenate the UHPSFC system to the ESI source of the mass spectrometer. As described elsewhere41, this interface consists of 2 serial tee unions that sequentially allows for connection of a make-up pump (ISM, Waters, Milford, MA, USA) and to split the effluent flow toward both MS and BPR. Two different designs of the splitflow interface were evaluated during method optimization. The first, which is the commercially-available Waters interface, consists of a 750 mm long, 50 µm I.D. PEEKsil restrictor

capillary connected to the top of the ESI probe. The probe contains a 236 mm long, 127 µm I.D. steel ESI emitter capillary. The fluidic connection between the PEEKsil capillary and the steel capillary is achieved by a user-installed PEEK zero-dead-volume (ZDV) union integrated to the top of the ESI probe. The second, which is a prototype Waters interface design, consists of a one-piece 750 mm long, 50 µm I.D. PEEKsil restrictor capillary restrictor permanently attached to 56 mm long, 127 µm I.D. steel ESI emitter capillary. There is no longer a fluidic connection at the top of the probe. Method development. Following the UHPSFC-ESIMS/MS path, different parameters were evaluated during the method development. First, six different stationary phase chemistries were tested for optimal selectivity, namely Viridis HSS C18 SB (C18), Viridis BEH (BEH), Viridis BEH 2Ethylpyridine (2-EP), Viridis CSH Fluoro-Phenyl (FP), Torus 1-Aminoanthracene (1-AA), and Torus Diol (Diol). All columns were purchased from Waters (Milford, MA, USA) in equivalent dimensions of 3.0 x 100 mm and packed with 1.7 µm particles except for Viridis HSS C18 SB which had 1.8 µm particles. All columns were rinsed and conditioned prior to column screening using 20 % (v/v) of MeOH in CO2. A 6 min scouting gradient of MeOH with 20 mM AmF in CO2 was carried out at 2 mL/min. The modifier proportion was kept at 2 % during a 0.5 min initial isocratic step, then linearly increased up to 20 % in 5 min and maintained at 20 % for a final isocratic step of 0.5 min. The gradient was followed by a 2 min re-equilibration step at 2 % of modifier. The effect of an addition of 2 % water to the organic modifier (v/v) was also assessed. MeOH, IpOH and a mixture of IpOH/heptane (3/7, v/v) were tested as MS make-up solvents at different flow rates (i.e. 100, 300 and 600 µL/min). The final separation was performed on the Viridis HSS C18 SB (C18) column using an optimized gradient of MeOH + 20 mM AmF + 2 % water (v/v) in CO2 (Supplementary Table S1). Liquid CO2 was pumped using a tandem head pump refrigerated at 13 °C and 2 °C for the accumulator and primary pump heads, respectively. The system pressure recorded at the pumps varied from 300 bar at the beginning to 340 bar at the end of the gradient. Column temperature and BPR pressure were set at 40 °C and 172 bar, respectively. The mixture of IpOH/heptane (3/7, v/v) at 100 µL/min was used as the MS make-up solvent and the prototype UHPSFC-ESI interface was selected. The optimal ESI parameters were set as follows: capillary voltage: +3 kV, ion source temperature: 150°C, desolvation gas flow rate: 650 L/h, desolvation temperature: 350 °C, nebulization gas pressure: 7 bar, cone gas flow rate: 150 L/h. The system was operated in SRM mode. Both collision energy and cone voltage were individually optimized for each SRM transition (Supplementary Table S-2). Argon (purity 99.999%) was used as collision gas at a flow rate of 0.2 mL/min. Due to the relative high endogenous concentration of α-tocopherol, the SRM method was developed using the second most abundant isotope in order to avoid detector saturation. Data acquisition and treatment. MassLynx 4.1 software suite (Waters, Milford, MA) was used to control the UHPSFCMS/MS system and to perform data acquisition. TargetLynx software package (Waters, Milford, MA) was used for automated peak integration while data processing was performed MS Office Excel software (Microsoft, Redmond, WA, USA).



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RESULTS AND DISCUSSION As we aimed at developing a fast and robust quantitative profiling of FSV and carotenoids in human plasma in a single assay for research, epidemiology, and routine applications, we opted for the automation and parallelization of the sample preparation while minimizing the required sample volume. A robotized SLE solution was used to multiplex the simultaneous extraction of 14 fat-soluble vitamins and carotenoids (chemical structures detailed in Figure S-1 in the supplementary material) from 200 µL of plasma. Subsequently, the plasma extracts were analyzed using a single UHPSFC-MS/MS method with a total run time of 8 minutes per sample including 2 minutes of re-equilibration. UHPSFC-MS/MS method development. Due to the presence of pairs of positional isomers, achieving suitable chromatographic resolution was mandatory to fulfill validation selectivity criteria. A column screening step was initiated in order to identify a stationary phase able to provide both suitable retention of the compounds of interest and at least a partial resolution of the critical pairs. Six UHPSFC dedicated columns namely, C18, BEH, 2-EP, FP, 1-AA, and Diol were tested with a fixed mobile phase condition consisting of a 5 min scouting gradient from 2 to 25 % of MeOH containing 20 mM AmF. Separation chromatograms are shown in supplementary material (Figure S-2). In spite of the variety of tested stationary phase chemistries, the elution order remained surprisingly comparable between columns. Punctual selectivity modifications affecting the resolution were however observed. Columns differed substantially in terms of achievable peak shape. Among the screened stationary phases, BEH, Diol, 2EP were unsatisfactory at first glance since it was not possible to properly elute vitamin K. Furthermore, unsuitable peak shape was observed for retinal on BEH and 2-EP columns while strong fronting was observed for some of the vitamin E isomers on BEH and Diol columns. Degraded peak shape for some of the vitamins E isomers was also observed with FP stationary phase. In addition, the FP column was not able to initiate the separation between β-tocopherol and γ-tocopherol critical pair. It is also worth mentioning that the FP stationary phase was surprisingly poorly retentive. Indeed it could have been expected that π-π interactions between the FP ligand and unsaturated bonds of the analytes would have contributed to better retention. Good retention and overall suitable peak shape were observed on both 1-AA and C18 stationary phases. The C18 column was identified as the most promising since it additionally provided the selectivity to separate all critical pairs. This column was therefore selected as the final support and the resolution was optimized through small and local adjustment of the gradient slope. Once the separation was optimized, its repeatability was assessed prior to initiating the validation process using multiple consecutive injections of the mixture of the 14 FSV and carotenoid standards. This preliminary test showed unexpected reduction in the compounds retention time (RT) after only a few dozen injections. This phenomenon was observed on 3 different batches of C18 stationary phases. This overall decrease in retention time could not be attributed to changes in mobile phase density generated by an increase in column pressure drop consecutive to premature column aging or frits clogging. Indeed, the observed system pressure, pump and oven temperature as well as BPR experimental pressure remained fairly constant. As hypothesized by Fairchild et al.42, the retention changes could be inherent to methyl silyl ether formation caused by a condensa-

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tion reaction between the stationary phase silanols and MeOH of the mobile phase. This reaction alters the surface polarity of the stationary phase causing a modification of the analyte RT. As it was reported elsewhere42-44, adjunction of a small proportion of water in the mobile phase (reversing condensation reaction equilibrium42) tend to stabilized the analytes RT. Accordingly, the mobile phase organic modifier was doped with 2 % of water. The addition of water required finer adjustments of the gradient slope to achieve proper resolution of the E vitamers critical pair as the presence of water in the mobile phase slightly modified the chromatographic interactions (Supplementary table S-1). The optimized stationary phase, mobile phase composition, and gradient profile resulted in excellent separation and peak shape for the 14 FSV and carotenoids (Figure 1). RT stability assessment was conducted again with the water-containing mobile phase. Significant improvement of RT stability was observed with less than 0.3% RSD over 150 injections. Both RT stability and separation resolution were maintained when the 14 FSV and carotenoids were spiked into human plasma matrix.

Figure 1. UHPSFC-MS/MS analysis of the 14 fat-soluble vitamins and carotenoids spiked in human plasma using the optimized mobile phase conditions and C18 stationary phase.

Make-up solvent selection. A significant and gradual decrease in MS-signal intensity over multiple injections of plasma was observed. This behavior culminated in the complete loss of ESI spray and thus MS-signal, after a prolonged analytical sequence of replicates. The loss of spray was consecutive to the clogging of the fixed restrictor capillary as its replacement enabled full recovery of spray and the MS signal. We hypothesized that this issue was due to the gradual precipitation of highly lipophilic matrix constituents previously extracted and concentrated by the SLE sample preparation. It is known that in SFC conditions, the CO2-based mobile phase displays good solvating property for lipophilic compounds as long as the fluid remains compressed. When hyphenated to MS, the mobile phase decompresses along the interface restrictor capillary. During decompression, the CO2 loses its solvating power, thereby relying entirely on the mobile phase modifier to keep species in solution. Precipitation can occur when species have limited solubility in the mobile phase modifier alone. Decreasing the polarity of the cosolvent could prevent this issue but would modify the separation selectivity. Alternatively, the make-up solvent can be optimized to increase the solubility of the lipophilic matrix constituents after CO2 decompression. We thus tested both IpOH and IpOH/heptane (3/7; v/v) as more lipophilic make up solvent alternatives. The latter mixture was tested as it corresponds to the sample diluent and also because, without the presence of IpOH, the MeOH based mobile phase modifier would not be miscible with pure heptane after the CO2 decompression. A new fixed restrictor capillary was mounted before each tested


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variation of the make-up fluid conditions. MS signal intensity was monitored for lutein, the most lipophilic compound of the set, over more than 150 consecutive injections of spiked plasma (Figure 2). In spite of the higher lipophilicity of IpOH, the lutein MS signal intensity decreased similarly when using MeOH as a make-up solvent. This observation tended to indicate the neat solvating power of the liquid effluent (modifier + make-up solvent) could not prevent the precipitation of the lipophilic constituents of the matrix that already started to gradually clog the fixed restrictor. In the case of the mixture of IpOH/heptane (3/7; v/v), the signal remains stable over the 150 injections (RSD 3.2%), which indicates that the precipitation was eliminated without any loss in sensitivity compared to the other tested make-up solvents. Under these conditions, the signal intensity was also observed to be about twice that which was observed with MeOH and IpOH make-up solvents. Although work is ongoing to explain this difference in sensitivity, the reduction of ESI droplet surface tension, consecutive to heptane adjunction, may explain the observed improvements of ESI ionization efficiency45. A second possible explanation may be due to the improved analyte transport (i.e. reduction in analyte precipitation) through the restrictor due to the net decrease in polarity after the addition of heptane. Such improvement in transport would be immediately evident as increased signal intensity.

Figure 2. Lutein MS-signal intensity follow-up over 150 consecutive injections of spiked human plasma using different makeup pump solvent delivered at a flow rate of 300 µL/min , i.e. MeOH (green squares, green dotted line), IpOH (red dots, red dotted line), and IpOH/Heptane (3/7, v/v) (blue triangles, blue dotted line).

After the optimal composition of the make-up fluid was selected, various makeup flow rates were evaluated. 100, 300, and 600 µL/min were screened against peak intensity and method robustness. Low make-up flow rates resulted in unstable ESI spray and irregular peak profiles. Such observations led to an effort to explore and improve the MS interface.

of peak profiles (Figure 3). The peak profile degradation randomly affected compounds all over the chromatogram from less retained vitamin K1 to lately eluting compounds such as lutein. Unlike the capillary clogging phenomenon described earlier, alterations of the peak profiles were observed with the IpOH/heptane (3/7; v/v) make-up fluid, thus, excluding a contribution from the plasma matrix itself. Using the split-flow UHPSFC-MS interface, the portion of mobile phase routed towards the ESI source through the restrictor capillary is under direct influence of the mobile phase flow rate, mobile phase composition, and the BPR pressure setting41. Once the mobile phase flow rate, column temperature and BPR pressure are set, only the viscosity of the mobile phase can affect the portion of the mobile phase directed to the MS through the restrictor capillary. The viscosity of the mobile phase is affected by changing the amount of modifier and through the addition of make-up fluid. Nevertheless, since the gradient program is relatively narrow, and the addition of the make-up solvent to the mobile phase further normalizes the change in split ratio inherent to gradient program, the portion of the mobile phase directed to the MS is relatively constant across the gradient. Therefore, it is unlikely that method conditions or changes in mobile phase composition could be responsible for the observed inconsistency in peak profile. One important factor to consider to maintain efficient analyte transport when employing a CO2-based mobile phase is to keep the latter in pressurized state for as long as possible. Decompressing far from the ion source causes density and solvating power drops as well as flow rate inconsistencies toward the ESI tip. Another important and related aspect to consider is the homogeneity of the mobile phase during the decompression process. Under high pressure, CO2 has sufficient density, and therefore solvating power, for complete miscibility with the other mobile phase components (i.e. cosolvent and make-up fluid). This high pressure state is obviously maintained along the whole chromatographic path towards the BPR. Sufficient pressure is also achieved at the inlet of the restrictor capillary that routes a portion of the effluent towards the MS. However, as the mobile phase flows through the restrictor, it gradually decompresses along the length. At some point, the CO2 density decreases to a point where it can no longer fully dissolve the other mobile phase components. The mobile phase becomes a heterogeneous flow of gas-phase CO2 and liquid droplets which have fallen out of solution. Akin to water drops on a window pane collecting in a fine rain, given enough time, the small droplets will coalesce into larger drops before rolling down the glass. Similarly, with enough post-decompression volume on the road towards to the ESI spray, the liquid component of the mobile phase will collect into larger drops within the heterogeneous flow. As the drops reach the end of the emitter, the resultant ESI spray plume randomly experiences visible and dramatic pulsing in the ion source (Video 1 in supplementary material), resulting in the inconsistent, spiked peak profile observed in Figure 3.

UHPSFC-MS interface robustness. Interfacing the compressed CO2-based SFC effluent to an atmospheric pressure ionization source such as ESI requires special attention. Particularly, the impact of the mobile phase compressibility on the transport of the analytes to the ion source has to be carefully considered38. When monitoring intra-day precision (RSD), the values obtained for most of compounds were already over the ± 15 % variability tolerance limits. A closer look at some chromatograms revealed unexpected and inconstant alterations



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Figure 3. A. Peaks spiking due to inconstant spray pulsing that occurred randomly. B. Observed peaks in absence of spray pulsing.

Figure 4. UHPSFC-MS interface design schematically detailing the fluidic differences between the commercially-available interface (A.) and the prototype interface (C.). The picture (B.) shows the fluidic domain of corresponding interfaces removed from the ESI probe assembly.

Spiked peak profiles can induce quantitation issues and will often resulting in large RSD values. Although altering the make-up fluid flow rate could help minimize spray pulsing, flow rate increase was not considered due the increased solvent consumption and to an observed decrease in sensitivity at high make-up flow rates. Another option consists of minimizing the postdecompression volume. In other words forcing the decompression to occur as close as possible to the ESI spray. Since the commercially available UHPSFC-MS interface (Figure 4A) contains considerable post-decompression volume (the 236 mm x 127 µm I.D. emitter capillary inside the ESI probe assembly), it promotes the spiked peak profiles and spray pulsing. To further provide evidence for the contribution of postdecompression volume to spray pulsing, the restrictor capillary was detached from the ESI probe. Without any additional volume at the outlet of the restrictor, the observed decompression of the mobile phase was very smooth and consistent and without any observable spray pulsing (Supplemental Video 2). Therefore, minimizing post-decompression volume in the system is very beneficial to ESI spray stability in UHPSFCMS. One opportunity for such an improvement in the current interface is to modify the ESI probe assembly itself. This probe assembly was originally designed for a relatively incompressible LC mobile phase. The commercially available interface employs a zero-dead-volume (ZDV) fluidic union for connecting the restrictor capillary to the emitter capillary. This type of union requires placement at the end of the probe due to its size. Accordingly, the large volume emitter capillary must extend along the entire length of the probe assembly. Alternatively, hardware modifications were performed as illustrated in Figure 4C. In this new prototype design, the ZDV PEEK connection at the top of the probe was eliminated. The fluidic connection between the restrictor capillary and the emitter capillary was performed with a Waters proprietary highpressure connection. The smaller outer diameter of this union compared to the conventional ZDV fluidic union allowed for placement of the restrictor inside the probe assembly. Accordingly, the necessary length of the emitter capillary could be reduced (56 mm x 127 µm I.D.). The short emitter capillary translates into much reduced post-decompression volume in the prototype interface and is still able to conduct capillary voltage to the emitter.

Although the mobile phase is fully decompressed at the point of spray with both interfaces, the reduced decompression volume of the prototype interface does not allow for the liquid droplets to collect into large drops as previously experienced. The prototype interface provided stable ESI spray (Video 3 in supplementary material), and consequently improved peak shapes. Subsequently, the enhanced ESI spray stability improved quantitation and peak repeatability metrics. Intra-day precision (RSD) showed values lower than 12 % for all the compounds, at both low and high concentration levels. Interestingly, the spray stability could be maintained even at reduced make-up pump flow rate with fused restrictor/emitter design prototype. A stable spray plume ensuring proper peak shape was still observed for a reduced make-up fluid flow rate of 100 µL/min over the entire separation gradient. This reduced flow rate provided optimal response without observed signal loss over multiple injections and was therefore selected as the make-up flow rate for the method. To further improve the UHPSFC-MS interface robustness, a restrictor rinse step was added to the method. During the isocratic hold at 20% B, the makeup flow rate was increased to 1.0 mL/min. This increase in flow was designed to flush the restrictor and remove any analyte or matrix material which may have precipitated during the separation. The makeup flow rate was returned to 0.1 mL/min for re-equilibration prior to the next injection. Validation. The method development allows a robust analytical procedure to quantify 14 FSV and carotenoids in human plasma. Plasma samples from a pool of donors were used as matrix to prepare calibration curves to test linearity. Since FSV and carotenoids are endogenous compounds, subsequent subtraction of the endogenous levels (measured on 6 nonspiked pooled plasma sample) from the calibration curves in the matrix is required during data treatment. Linearity, LOD and LOQ. The linearity of the method was determined with calibration curve in the matrix spiking ten levels per compound in triplicate. Results are reported in supplementary material (Table S-3). The concentration ranges were: 20 ng/mL to 1400 ng/mL for lutein, zeaxanthin, αtocotrienol, γ-tocotrienol, δ-tocotrienol, β-tocopherol, γtocopherol, δ-tocopherol, 25-hydroxyvitamin D2, and 25hydroxyvitamin D3, 0.2 ng/mL to 14 ng/mL for vitamin K1,


Page 7 of 8

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and 1600 ng/mL to 112000 ng/mL for α-tocopherol. Regression analysis provided R2 value above 0.999 for all the compounds. The LOD and LOQ were calculated in solvent due to the endogenous levels of FSV and carotenoids in plasma. LOD and LOQ values for each compound were defined with a signal-to-noise ratio of at least 3 and 10, respectively. For αtocopherol, the second most abundant isotope was used in the SRM method to avoid detector saturation induced by endogenous concentration.

Trueness and Precision. Intra-day and inter-day variations were calculated over a six-day precision test. Three different concentration levels were tested (low, medium, high) using four replicates (QC) per level. For each day, calibration curve and QC samples were run together. The obtained trueness and precision values comply with FDA recommendations for validation (i.e. trueness in the range of 100 ± 20 % and RSD values below 15 %). The data of the precision test are reported in Table 1. All the QC samples showed trueness and precision values between 85-115 % and 2-14 %, respectively.

Table 1. Accuracy and Precision of the Assay. Compound

Low level [ng/mL]

Intraday (RSD)

Interday (RSD)

Trueness

Medium level [ng/mL]

50

6.5 %

14.4 %

100.7 %

2

8.9 %

9.7 %

96.5 %

α-tocopherol

1600

4.8 %

8.9 %

α-tocotrienol

200

4.3 %

10.4 %

β-tocopherol

200

6.8 %

13.0 %

97.3 %

400

8.2 %

14.0 %

γ-tocopherol

200

9.0 %

10.5 %

97.7 %

400

10.3 %

12.4 %

δ-tocopherol

200

3.7 %

9.2 %

97.6 %

400

5.3 %

7.8 %

γ-tocotrienol

200

5.5 %

6.2 %

93.6 %

400

5.2 %

δ-tocotrienol

200

5.2 %

10.8 %

92.0 %

400

5.7 %

25-Hydroxyvitamin D2

50

7.1 %

7.3 %

100.0 %

400

7.4 %

25-Hydroxyvitamin D3

50

9.0 %

9.0 %

102.0 %

400

β-cryptoxanthin

200

11.9 %

12.3 %

104.7 %

Lutein

400

5.0 %

10.0 %

Zeaxanthin

400

5.5 %

8.4 %

Retinal Vitamin K1

Intraday (RSD)

Interday (RSD)

Trueness

High level [ng/mL]

200

6.6 %

4

3.2 %

94.8 %

32000

104.5 %

400

Intraday (RSD)

Interday (RSD)

Trueness

12.7 %

87.7 %

800

3.9 %

8.9 %

98.7 %

5.4 %

99.7 %

8

2.7 %

3.9 %

102.2 %

4.8 %

5.5 %

104.6 %

64000

5.3 %

7.3 %

107.8 %

6.8 %

12.5 %

99.9 %

800

5.2 %

13.8 %

106.9 %

97.7 %

800

3.9 %

6.2 %

99.1 %

104.7 %

800

6.2 %

8.0 %

104.4 %

100.7 %

1200

6.5 %

8.4 %

111.8 %

7.5 %

95.0 %

800

4.8 %

5.0 %

99.8 %

10.7 %

94.9 %

800

5.0 %

13.5 %

103.3 %

7.8 %

100.2 %

800

5.6 %

6.0 %

101.9 %

6.8 %

6.6 %

101.8 %

800

5.4 %

6.3 %

103.6 %

400

9.6 %

9.4 %

109.8 %

1200

5.9 %

7.1 %

104.9 %

107.5 %

800

4.4 %

11.9 %

111.8 %

1200

4.0 %

5.2 %

105.8 %

109.7 %

800

4.4 %

8.9 %

110.2 %

1200

3.6 %

6.4 %

100.2 %

Stability. The stock solution of the standards (including IS) were stable for 1 month at -80 °C. All working solutions were prepared fresh on a weekly basis and stored at -80 °C. We observed sample instability 24 hours after extraction. We hypothesize that such instability is due to the evaporation of the IpOH/heptane diluent which is used to reconstitute the sample before the injection. The increased response over time is consistent with diluent evaporation and seems to support this assumption.

decompression leading to satisfactory robustness. The reported methodology could be further expanded to other liposoluble nutrients and micronutrients for the comprehensive analysis of body fluids.

CONCLUSIONS

O'Brien of the Waters Wilmslow Mechanical Design team for the development of the ESI probe and John Corthésy from Nestlé institute of health sciences.

The present developments enable fully validated fast quantitation of 14 FSV and carotenoids in human plasma. The proposed analytical workflow was simplified and automated with the goal to improve cost-efficiency, sustainability, and sample volume requirements for both research and routine utilizations. The improved UHPSFC-ESI-MS/MS method offers an efficient alternative to LC for the high-throughput and robust analysis of chemically sensitive FSV and carotenoid compounds. The method enables the separation of structurally similar isomers such as lutein and zeaxanthin and the family of tocopherols in 8 minutes using a relatively small volume of organic solvent. UHPSFC-MS interface improvements were developed to better manage the mobile phase

ACKNOWLEDGMENT Authors acknowledge R. Plumb, J. Langridge, S. Canarelli, and M. Rentsch from Waters Corporation for supporting the development of the UHPSFC-ESI-MS/MS method, I. Trivett and S.

Supporting Information The above mentioned supplementary figures, tables and videos are available free of charge via the Internet at http://pubs.acs.org.”

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