Field of Invention

[0001] The present invention relates to a high-throughput method for a rapid screening platform for 25-hydroxyvitamin D (25OH-D) and related metabolites. Specifically, the present invention uses blood specimens for the determination of vitamin D status based on direct infusiontandem mass spectrometry (DI-MS/MS) following click derivatization using 2-nitrosopyridine.

Background of the Invention

[0002] Vitamin D, including cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2), represents a group of fat-soluble prohormones essential for human health. Vitamin D is primarily derived from UVB solar radiation conversion of 7-deoxycholesterol in the skin, diet, and supplements. It is subsequently metabolized in the liver into 25-hydroxyvitamin D (25OH-D or calcidiol) that serves as the significant and stable circulating reservoir of vitamin D before its activation into 1,25 dihydroxy vitamin D (calcitriol) and various other hydroxylated metabolites and their isomers. Other factors can also impact vitamin D nutritional status, including underlying medical conditions, abnormal fat absorption, aging, obesity, dark skin pigmentation, drug intake, and lifestyle such as prolonged indoor activities.

[0003] Vitamin D deficiency is known to impair calcium homeostasis and bone mineralization in nutritional rickets and osteomalacia, while also contributing to several extra- skeletal pathologies relevant to cardiac, respiratory, neurological, and immune system dysfunction given the widespread expression of the vitamin D receptor in various tissues and blood cells. Also, the seasonal and latitude dependence on vitamin D deficiency has long been implicated in epidemic influenza with a periodic wintertime excess in infection and mortality. Similarly, low circulating 25OH-D concentrations are associated with a greater risk of SARS-CoV-2 disease, severe illness, and mortality during the COVID-19 pandemic, given its mechanisms in regulating innate and adaptive immune responses. This is concerning given that a third and more than two-thirds of Canadians are estimated to be vitamin D deficient (25OH-D < 50 nmol/L) and insufficient (25OH- D< 75-80 nmol/L), respectively, which is prevalent among older persons with dementia in longterm care facilities. Thus, rapid screening platforms that enable reliable assessment of vitamin D nutritional status in high-risk populations are needed to guide optimal prophylactic and early treatment strategies to prevent hospitalization from severe COVID-19 disease.

[0004] The clinical significance of vitamin D deficiency has increased demand for reliable tests for 25OH-D determination in blood specimens, regarded as the sum of 25OH-D2 and 25OH- D3. Competitive protein binding assays, radioimmunoassays, and chemiluminescence immunoassays have been established to analyze 25OH-D and other hydroxylated vitamin D metabolites in serum or plasma samples. However, the cross-reactivity and reproducibility of commercial immunoassays used in clinical laboratories vary significantly between manufacturers, complicating the development of consensus 25OH-D cut-off concentrations to define vitamin D nutritional status. For these reasons, liquid chromatography with tandem mass spectrometry (LC- MS/MS) is recognized as the gold standard for quantitative 25OH-D determination due to its excellent selectivity, lower detection limits, and improved precision immunoassays. Yet, different column types, elution conditions, ion sources, and sample workup protocols with or without chemical derivatization have been developed to identify bioactive metabolites of 25OH-D that do not always resolve bioactive metabolites from metabolites which are less active metabolites (such as the 3-epimer of 25OH-D3). Longer run times (>10 min) in LC-MS/MS are often needed to separate 25-OH-D3 from lower levels of its 3-epi-25OH-D3 isomer. In addition, LC-MS/MS is more costly and thus not practical for high-throughput use.

[0005] An improved method of vitamin D screening is desirable to overcome disadvantages of current methods.

Summary of the Invention

[0006] The present invention provides a novel screening method for vitamin D, wherein a direct infusion-tandem mass spectrometry (DI)-MS/MS protocol is used to detect derivatives of vitamin D to enhance the ionization efficiency of labelled 25OH-D cycloadducts in serum or plasma extracts. This method enables high throughput screening of vitamin D status that is not practical when relying on LC-MS/MS while offering better analytical and diagnostic performance than that provided by commercial immunoassays.

[0007] The present invention, in one aspect, generally relates to a method of screening for vitamin D status from a biological sample comprising the steps of: a) obtaining a biological sample; b) derivatizing vitamin D and metabolites within the sample; and c) acquiring and processing DI-MS/MS data regarding derivatized vitamin D and metabolites thereof within the sample to determine vitamin D status of the sample.

[0008] In another aspect of the invention, a method of determining vitamin D status of a biological sample comprising: i) subjecting the biological sample to extraction; ii) derivatizing vitamin D and metabolites within the sample to form detectable vitamin D derivatives; iii) subjecting the derivatized sample to back extraction under alkaline conditions; and iv) analyzing the derivatized sample using direct infusion-tandem mass spectrometry (DI-MS/MS) under suitable conditions to detect the vitamin D derivatives in the sample to provide the vitamin D status of the sample.

[0009] In a further aspect of the invention, a method of preparing a biological sample for analysis of Vitamin D status comprising the steps of: i) subjecting the biological sample to a first extraction; ii) derivatizing vitamin D and metabolites thereof within the sample to form detectable vitamin D derivatives; and iii) subjecting the derivatized sample to back extraction under alkaline conditions.

[0010] These and other aspects of the invention will become apparent from the detailed description and Figures which follow.

Brief Description of the Drawings

[0011] Figure 1 shows the collisional-induced dissociation experiments (at 10 V) for MS/MS characterization of: (A) 1,1 -dimethyl -N-(pyridin-2-yl)-4-sulfanimine (intermediate) that reveals several significant fragment ions, and (B) the final product, 2-nitrosopyridine, used as a reagent for chemical derivatization of 25OH-D metabolites via a Diels-Alder reaction, which generated a distinctive fragment ion following a neutral loss of NO (m/z 29.9979).

[0012] Figure 2 shows that deleterious impact of matrix-induced ion suppression effects during DI-MS/MS was reduced by performing a back extraction under alkaline conditions (500 ji/L hexane, 400 j L water, 25 j L ammonium hydroxide) following chemical derivatization of Sigma serum samples (50 j L acetonitrile) to remove excess reagent and by-products from derivatized vitamin D cycloadducts. (A) An extracted ion electropherogram (EIE) of purine (a reference mass continuously infused into the ion spray) shows a significantly greater (13.3-fold vs. 2.1-fold) reduction in signal when no back extraction is used. EIEs for (B) 25OH-D3 adduct and (C) d6-25OH-D3 adduct both consistently show greater sensitivity (~ 8-fold) after performing a back extraction to reduce ion suppression before DI-MS/MS.

[0013] Figure 3 shows (A) optimization of reaction conditions for quantitative adduct formation between 2-NO-Pyr and 25OH-D3. Reaction time (30 min) and solvent composition (acetonitrile) were kept constant, while temperature and reagent concentration was varied. Each optimization experiment was performed in triplicate. (B) Chemical derivatization at 60 °C using 10 mmol/L 2-NO-Pyr resulted in > 95% conversion with an enhanced ion response (i.e., slope) for the 25OH-D3 cycloadduct, which was more than 10-fold greater than underivatized 25OH-D3 as control. All experiments were performed in triplicate with error bars representing ± 1 SD. Parallel reaction monitoring (PRM) transitions on a quadrupole-time-of-flight (Q-TOF) mass spectrometer were optimized for each compound using the same ion source conditions. Namely, the derivatized 25OH-D3 adduct (m/z 509.4 — m/z 227.1179 at 30 V) compared to the unlabelled 25OH-D3 (m/z 401.3 — 383.3308 at 5.0 V; neutral loss of FEO).

[0014] Figure 4 shows a schematic depicting the (A) rapid screening of vitamin D nutritional status by DI-MS/MS following 2-NO-Pyr click derivatization and liquid-phase extraction of blood specimens using a CE system to automate sample injection and infusion into coaxial sheath liquid interface with a total duty cycle of 3.3 min/sample. (B) PRM (parallel reaction monitoring) measured product ions following collision-induced dissociation of labelled 25OH-D cycloadducts under positive ion mode detection. (C) Reference serum 25OH-D3 control chart. (D) Acceptable intraday precision (mean CV = 10.8%) was achieved with continuous analysis of standard serum extracts (n=412) and blank (n=20) samples over a 24 hr period after data smoothing and normalization of ion responses to a stable isotope internal standard. Good linearity (R ² > 0.99) and precision (mean CV < 10%) was achieved for calibrants over a wide dynamic range for (E) 25OH-D3 and (F) 25OH-D2. (G) Spike and recovery studies in standard serum samples confirmed acceptable method accuracy for 25OH-D3 (103 ± 12) % and 25OH-D2 (101 ± 12) % quantification at different concentration levels (15, 30, and 45 nmol/L). [0015] Figure 5 shows (A) extracted ion electropherogram of d6-25OH-D3 fragment ion (m/z 515.4 — 227.1179) under steady-state conditions from a large (~ 1.1 pL) sample plug allowing for the acquisition of about 13 data points shown as blue circles. Raw data is Gaussian smoothed before peak integration for quantitative 25OH-D determination with serum or plasma 25OH-D3 and 25OH-D3 responses normalized to d6-25OH-D3. In this case, capillary electrophoresis is used for sample introduction for direct programming infusion of blood extracts, calibrants or blanks into a coaxial sheath liquid interface coupled to a Q-TOF-MS system with parallel reaction monitoring under positive ion mode. Importantly, no separation or voltage application was applied in CE during the sample loading and infusion period. (B) Series of calibrant solutions used for 25OH-D3 quantification after data smoothing over a wide linear dynamic range (5-400 nmol/L) relative to blank (0 nmol/L) by DI-MS/MS. In this case, CE is used as a sample introduction device for programming direct infusion of blood extracts, calibrants or blanks into a coaxial sheath liquid interface coupled to a Q-TOF-MS system with PRM under positive ion mode. No separation or voltage application is applied in CE during the sample loading and infusion period.

[0016] Figure 6 shows an inter-day reproducibility evaluation of the DI-MS/MS method based on measuring ten standard Sigma serum extracts sequentially, followed by a blank and a 6- point calibration curve. This block of runs was repeated four more times each day and then reanalyzed over two other days. Each day, a new fused-silica capillary was prepared, the ion source was cleaned, and the MS instrument tuned. A control chart highlights the reliability of the technique as no serum extract sample analyzed exceeded action limits (± 3 s) with a mean CV of 10.5% (n = 150) over three different days.

[0017] Figure 7 shows the long-term chemical stability of 25OH-D from plasma extracts using DEQAS reference samples (566-580), demonstrating no significant Di els- Alder cycloadduct product degradation following two repeat free-thaw cycles (overall CV = 11.0%) with storage in a -80 °C freezer and subsequent thawing at room temperature.

[0018] Figure 8 shows an inter-method comparison between DI-MS/MS and validated LC- MS/MS methods for 25OH-D quantification using reference blood samples from DEQAS and NIST (n=18). Overall, a good mutual agreement was demonstrated as reflected by (A) a Passing- Bablok regression showing a slope of 1.17 within 95% confidence limits of the line of equality, and a (B) a Bland-Altman % difference plot with a mean bias of 7.8% with only one outlier exceeding agreement limits. (C) A Mountain plot overlay highlights the greater bias of a commercial FDA-approved chemiluminescence immunoassay system (Qualigen Fastpack® Immunoanalyzer) relative to DI-MS/MS when reporting circulating 25OH-D concentrations from reference blood samples (n=18) independently measured by LC-MS/MS. (D) An empirical cumulative distribution plot overlay of plasma 25OH-D concentrations measured in a cohort of hospitalized pediatric patients (n=30) highlights the impact of method bias when screening for vitamin D nutritional status. A greater fraction of patients are identified as being vitamin D deficient (25OHD < 50 nmol/L) when screening is performed by DI-MS/MS (43%) as compared to the commercial immunoassay (16.7%) that is prone to positive bias (overestimates true 25OH- D concentration), impacting clinical treatment decisions due to misclassification.

[0019] Figure 9 shows the mean bias of the DI-MS/MS method relative to LC-MS/MS is reduced to only 1.5% when accounting for 3-e/?z-25OH-D3 concentrations reported in the reference blood samples. Overall, the methods show excellent mutual agreement, suggesting that 3-epi-25OH-D3 is the major contributing factor for concentration differences measured between the two independent ways when chromatographic separations were not performed.

[0020] Figure 10 shows an inter-method comparison for quantifying 25OH-D concentrations from reference plasma/serum samples (DEQAS, 77=15; NIST, 77=3) between a commercial FDA-approved immunoassay system (Qualigen Fastpack® Immunoanalyzer) and LC-MS/MS. A Bland-Altman % difference plot confirmed a mean positive bias of 13% when measuring 25OH-D by immunoassay compared to LC-MS/MS given antibody cross-reactivity to 3-e/?z-25OH-D3 and other hydroxylated vitamin D metabolites.

[0021] Figure 11 shows the accuracy of the commercial immunoassay system (Qualigen Fastpack® Immunoanalyzer) in measuring 25OH-D as compared to DI-MS/MS in 4 reference serum samples from NIST 972a (levels 1 - 4). General agreement for serum 25OH-D concentrations with reference LC-MS/MS method was acceptable with a consistently higher average bias (~ 12%) for immunoassay compared to DI-MS/MS except the NIST reference sample enriched with 3-e/?z-25OH-D. The level 4 sample was fortified with 3-e/?z-25OH-D3 (64.8 nmol/L relative to 73.4 nmol/L 25OH-D3), which had a much higher positive bias (+42%) than anticipated indicative of antibody cross-reactivity against this stereoisomer (although rated as only as 7.8% cross-reactivity at 1000 nmol/L tested by the manufacturer; Qualigen FastPack® IP Vitamin D Immunoassay Kit Complete), which is typically present at low nanomolar levels (~ 7 nmol/L). The DI-MS/MS assay measures a total 25OH-D that, is the sum of 25OH-D3 and 3-e/?z-25-OHD together with 25OH-D2, thus exhibiting the most significant bias in this reference sample.

[0022] Figure 12 shows a pooled plasma quality control (QC) sample was analyzed over ten months before screening pediatric patient samples. During this period, five different lots with IP Vitamin D Kit Complete FastPack® test pouches were used intermittently by the commercial CLIA system. A control chart (A) shows variation in the QC samples from singlicate readings, where results are shown by lots. Overall precision was suboptimal (18%), with a notable difference between lots. (B) Box plots highlight batch effects as confirmed by a single factor ANOVA, where an inter-lot CV of 16% was measured. Batch information (lot number / first date used / last date used) were as follows: Batch 1 (2003028-3P / Jan-11-21 / Feb-8-21), Batch 2 (2009014-1P / Feb- 10-21 / Mar-9-21), Batch 3 (2009014-2PR / Mar- 16-21 / July- 12-21), Batch 4 (2009014-4PR / July-15-21 / Aug-25-21), and Batch 5 (2103013-2P / Sep-5-21 / Nov-16-21).

Detailed Description of the Invention

[0023 ] The present invention relates to a novel high-throughput method for the quantitative determination of vitamin D metabolites in biological specimens based on direct infusion-tandem mass spectrometry (DI-MS/MS) that forgoes the need for chromatographic separation.

[0024] The present method comprises: i) subjecting the biological sample to extraction to remove interferents; ii) derivatizeing vitamin D and metabolites within the sample to form detectable vitamin D derivatives; iii) subjecting the derivatized sample to back extraction; and iv) analyzing the derivatized sample using direct infusion-tandem mass spectrometry (DI-MS/MS) under suitable conditions to detect the vitamin D derivatives in the sample and provide the vitamin D status of the sample.

[0025] The biological sample to be analyzed using the present method include biological fluids such as whole blood, plasma, serum, dried blood spots, cerebrospinal fluid, saliva, sweat and urine; as well as tissue samples such as, but not limited to, the skin, hair, adipose tissue, immune cells, parathyroid gland, liver and kidney tissue. The biological sample may be obtained using known techniques based on the selected sample. Preferred biological samples are whole blood, plasma, serum and dried blood spots. As will be appreciated by one of skill in the art, tissue and cell samples may be subjected to a pre-processing step such as centrifugation to remove undesirable solids therefrom prior to further processing.

[0026] Once obtained, the biological sample is subjected to liquid extraction to remove interferences, such as proteins, lipids and carbohydrates, from the sample, and to release vitamin D from components within the sample, for analysis by DI-MS/MS. Interf erents such as proteins are removed from the sample by precipitation followed by centrifugation. Proteins and the like may be precipitated using an effective precipitating agent such as an organic solvent, e.g. acetonitrile, methyl-tert-butyl ether, hexane, methanol, acetone, chloroform, and the like. The precipitated sample is then subjected to centrifugation under conditions sufficient to form a protein pellet, e.g. about 5 minutes at 10,000g. The resulting supernatant is then subjected to extraction to remove other interferents to provide a concentrated vitamin D-containing layer or sample as supernatant, for example, using an organic non-polar solvent (e.g. an alkane such as pentane, hexane or heptane, an aromatic such as benzene or toluene, ether, diethyl ether, tert-butyl ethyl ether, ethyl acetate, methylene chloride, and the like) under acidic conditions (e.g. in the presence of a mineral acid such as hydrochloric, nitric or sulfuric acid). The organic non-polar solvent layer or extraction layer containing vitamin D and/or metabolites thereof is then removed from the pellet.

[0027] The vitamin D-containing extract is subj ected to a reaction to derivatize the vitamin D within the sample to form derivatives of vitamin or metabolites thereof which are readily detected within the sample. Vitamin D is derivatized in a manner that yields detectable derivatives, for example, derivatives which can readily be ionized and which can be detected at with sufficient sensitivity. In one embodiment, the sample is derivatized with a dienophile using the Diels- Alder reaction to form a substituted cyclohexene derivative by reaction of the dienophile with the conjugated diene of the vitamin D. The term “vitamin D” or “25OH-D” is used herein to refer to both vitamin D2 (25OH-D2) and vitamin D3 (25OH-D3), and metabolites thereof such as 25- hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D, 1,24,25-trihydroxyvitamin D3, and sulfate or glucuronide conjugates of vitamin D metabolites such as the 25-hydroxyvitamin D3 3-sulfate and 25-hydroxyvitamin D3 3-glucuronide conjugates. The derivatization reaction is conducted under conditions appropriate for a Diels- Alder reaction, in the presence of an organic solvent and application of heat, e.g. 55-65 °C, for an appropriate period of time.

[0028] Dienophiles or Cookson-type reagents suitable for use to derivatize vitamin D and metabolites thereof based on their s-cis-diene structure include, but are not limited to, 2- nitrospyridine (PyrNO), 1, 2, 4-triazoline-3, 5-dione (TAD), 4-phenyl-l, 2, 4-triazole-3, 5-dione (PTAD), as well as various substituted TAD analogs (e.g. DMEQ-TAD, DAPTAD, Ampliflex™ Diene, and SecoSET™ commercial reagents). Other analogs of PyrNO may also be used as viable reagents, such as 4-chloro-2-nitrosopyridine, 4-nitrosoquinoline and 2,2,3 -trimethyl- 1- nitrosoquinoline.

[0029] Following derivatization of the sample, and in particular vitamin D and metabolites thereof, back extrusion is then utilized as a final step to render the derivatized sample appropriate for analysis by DI-MS/MS. Back extraction is conducted using an organic non-polar solvent under alkaline conditions (e.g. in the presence of sodium or ammonium hydroxide, sodium bicarbonate and the like) to remove excess reagent (e.g. dienophile) and by-products of the reaction (e.g. pyridine) from derivatized vitamin D cycloadducts. The inclusion of a back extraction step in the present method surprisingly has been found to significantly reduce ion suppression to yield an enhanced signal and greater sensitivity (e.g. greater than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold or greater sensitivities) than the signal achieved without employing a back extraction step in the method. In an embodiment, sensitivity is increased by about 8-fold greater than the sensitivity of the signal achieved without employing a back extraction step in the method. The provision of enhanced signal sensitivity is important when performing DI-MS/MS with electrospray ionization without chromatographic separation.

[0030] The purified vitamin D derivatives are then advantageously analyzed using DI- MS/MS, without the need to first conduct separation of the sample using, for example, liquid chromatography. Any DI-MS/MS instrument may be used in the method as will be appreciated by one of skill in the art. For example, any device allowing for direct infusion or flow injection such as classic syringe/infusion pump, chip-based direct infusion with nanospray ionization, and an LC system with an injector may be used. Any type of direct infusion-MS interface may be used including standard electrospray ionization, or nanoflow or low-flow electrospray interfaces for greater sensitivity. Further, any tandem MS/MS analyzer or device may be used, including a triple quadrupole (e.g., multiple reaction monitoring, neutral loss scanning etc.), quadrupole-time-of- flight and quadrupole-orbitrap systems from various manufacturers. Further, additional selectivity for resolving vitamin D metabolite isomers (e.g., 3-e/?z-25-OH-D3) can be achieved in a high throughput manner by coupling ion mobility spectrometry prior to DI-MS/MS. Operating conditions are selected for the DI-MS/MS analysis of vitamin D metabolites based on the DI- MS/MS analyzer used, the capillary dimensions, the instrumental configuration, and the CE-MS interface.

[0031] The use of DI-MS/MS provides a more efficient analysis of vitamin derivatives in a sample in that it is quicker and less costly than methods which employ the current LC-MS/MS gold standard method, rendering it appropriate for high-throughput use. It also provides improved selectivity and better reproducibility without batch effects as compared to immunoassay methods.

[0032] Embodiments of the invention are described in the following specific example which is not to be construed as limiting.

Example 1: A DI-MS/MS Protocol for Rapid Screenins of Vitamin D

[0033] The following describes the development of a high-throughput method of Vitamin D screening in accordance with an aspect of the invention.

[0034] Reference Serum Samples and Plasma Collection from Critically III Children - Reference material NIST 972a consisting of human serum samples (L1-L4) with certified concentrations for vitamin D metabolites were purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, ML, USA). Human serum samples with reference values reported for vitamin D metabolites (566 to 580) as measured by a validated LC-MS/MS protocol were also purchased from the Vitamin D External Quality Assessment Scheme (DEQAS, London, UK). Also, plasma samples from critically ill pediatric patients at McMaster Children’s Hospital (from 02/2021 until 12/2021) were screened for vitamin D deficiency, which was approved by Clinical Trials Ontario (#1761). Blood samples were collected in EDTA coated tubes and centrifuged at 2000 g for 20 min at 4 C. The supernatant was transferred to a new uncoated line and stored frozen at -80 °C before analysis. [0035] Chemicals, Reagents and Reference Serum Samples - Reagents for sample preparation and sample infusion include UHPLC-MS grade methanol and acetonitrile, hexane (distilled in glass), and reagent grade concentrated hydrochloric acid from Caledon Inc. (Georgetown, ON, Canada). UHPLC-MS grade water was purchased from Honeywell Inc. (Muskegon, MI, USA). ACS reagent grade ammonium hydroxide and vitamin D standards including 25OH-D3 (250 pmol/L) in ethanol, 25OH-D2 (125 pmol/L) in ethanol and d6-25OH- D3 (125 pmol/L) in ethanol were purchased from Sigma Aldrich (Mississauga, ON, Canada). All other reagents were purchased from Sigma Aldrich unless otherwise noted.

[0036] Synthesis of 2-Nitrosopyridine as Derivatization Agent - A two-step synthetic procedure was used to prepare 2-nitrosopyridine (2-NO-Pyr) that serves as a click derivatization reagent to react selectively with 25OH-D metabolites to introduce a positive charge via a Diels- Alder reaction as previously described (Wan et al. 2017. J Lipid Res 58: 798-808). Briefly, 2- aminopyridine (3.95 g, 42 mmol, one equiv.) was transferred to a 500 mL round bottom flask (RBF) and dissolved in 40 mL of di chloromethane (DCM). To this, dimethyl sulfide (3.4 mL, 46 mmol, 1.1 equiv.) was added, and the RBF was placed in an ice bath. A solution of N- chlorosuccinimide (5.80 g, 42 mmol, one equiv.) in 100 mL DCM was added dropwise over one hour. The reaction was stirred for one hour on ice and then an additional hour at room temperature. In DCM, sodium methoxide (4.0 g, 74 mmol, 1.8 equiv.) was added and stirred for 10 minutes. Then 50 mL of water was added and stirred for an additional hour. The aqueous and organic layers were then separated, and the aqueous layer was washed twice with 30 mL of DCM. The combined organic solution was dried with sodium sulfate and then evaporated with a steady stream of nitrogen to produce an orange precipitate. Recrystallization from diethyl ether gave cream- coloured crystals (4.6 g, 69% yield).

[0037] MS/MS was performed at 10 V while monitoring all fragments between m/z 20 and 155, where several diagnostic fragments’ ions were detected for 1,1 -dimethyl -N-(pyridin-2-yl)-4- sulfanimine (Figure 1 A). 1H NMR (600 MHz, CDC13) 8 7.99 (d, J = 5 Hz, 1H), 7.32 (t, J = 8 Hz, 1H), 6.67 (d, J = 8 Hz, 1H), 6.47 (t, J = 6 Hz, 1H), 2.72 (s, 6H). 13C NMR (151 MHz, CDC13) 6 165.57, 147.21, 136.89, 114.27, 111.59, 33.91.

[0038] Next, m-chloroperoxybenzoic acid (6.4 g, 37 mmol, 1.7 equiv.) was dissolved in 150 mL of DCM and cooled on an ice bath. The intermediate from step 1 (3.4 g, 22 mmol, one equiv.) in 30 mL of DCM was added and stirred for 90 minutes, causing the yellow solution to turn green before returning to yellow. Then, 2 mL of dimethyl sulfide was added, and the solution was stirred for an additional 30 min. The contents of the round bottom flask were then transferred to a separatory funnel and washed with a saturated sodium bicarbonate solution. The organic layer was then washed twice more with 20 mL of water. The organic layer was then dried with sodium sulfate and evaporated with gentle nitrogen steam to give a dark orange precipitate. The precipitate was recrystallized three times from ethanol to give bright yellow needles (0.77 g, 32% yield). MS/MS was performed on a Q-TOF system using a collision energy of 10 V while monitoring all fragments between m/z 20 and 109, where only a single major fragment ion formed (m/z 79.0147) following a neutral loss (m/z 30) of NO (Figure IB). 1H NMR (600 MHz, CDC13) 6 8.02 (d, J = 5.1 Hz, 1H), 7.94 (t, J = 7.9 Hz, 1H), 7.88 (d, 8.1 Hz, 1H), 7.31 (m, 1H). 13C NMR (151 MHz, CDCh) 6 155.88, 147.08, 139.44, 125.69, 118.93.

[0039] Commercial Chemiluminescence Immunoassay for Vitamin D Screening - Vitamin

D screening of blood specimens was performed by a quantitative chemiluminescence immunoassay using the FastPack® Analyzer with IP Vitamin D Kit Complete test pouches by procedures from Qualigen Therapeutics Inc. (Carlsbad, CA, USA), which SI-4 included calibrators and controls. Also, internal quality control (QC) specimens were used to monitor the commercial immunoassay's long-term performance using pooled plasma samples collected from pediatric patients at CHEO. Briefly, 100 pL of plasma sample was transferred to a vial containing 200 pL of supplied pretreatment buffer and then inverted five times. 100 pL of this solution was introduced into the injector port of the FastPack® test pouch using a micropipette, which was then placed inside a Fastpack® IP instrument for automated analysis taking about 11 min each sample. All measurements were performed in duplicate or triplicate (for borderline vitamin D deficiency ranging from 45 to 55 nmol/L), and two QC runs with calibration were performed each month. This results in a total analysis time for immunoassay measurements of about 45 min to 60 min per sample and QCs. Results (concentration, date, and time) were printed onto the FastPack sticker containing the lot number, then peeled off and stored in a binder. The limit of detection (LOD), the limit of quantification (LOQ) and upper limit of linearity for the commercial immunoassay is reported by the manufacturer as 15.5 nmol/L, 32.2 nmol/L, and 375 nmol/L, respectively, with cross-reactivity noted for other hydroxylated vitamin D metabolites and negative bias when analyzing lipemic blood samples (see Qualigen. FastPack IP System - Procedure Manual. https://www.qualigeninc.com/wp-content/uploads/2019/03/65000 159.pdf (accessed Dec 10, 2021).

[0040] Liquid-Phase Extraction and Chemical Derivatization for 25OH-D Analysis by DI-

MS/MS - 32 /zL of 1.0 /zmol/L d6-25OH-D3 in ethanol was added to a glass amber vial followed by a 50 pL aliquot of thawed serum or plasma. Protein precipitation was initiated by adding 200 /IL of methanol and 50 pL of water, followed by shaking for 5 min. The solution was then centrifuged for 5 min at 10,000 g under 4 °C, and the supernatant was transferred to a new glass amber vial. 200 L of water, 25 L of 1.0 M HC1, and 500 L of hexane were added to this solution. Shaking was then performed for 5 min followed by centrifugation for 5 min at 10,000 g under 4 °C. The six upper hexane layer was transferred to a new amber glass vial and dried with a steady stream of nitrogen at room temperature. Then, 50 pL of a ten mmol/L 2-NO-Pyr solution in acetonitrile was added, and the vials were vortexed. A hot block was used to heat the reaction at 60 °C for 30 min. Derivatized plasma or serum extracts were then placed in the fridge to cool, after which 400 pL of water, 25 pL of ammonium hydroxide, and 500 pL of hexane were added. Shaking was performed for 5 min followed by centrifuging for 5 min at 10,000 g under 4 °C. The upper hexane layer was transferred to a new amber glass vial and dried under a nitrogen flow at room temperature. Lastly, dried extracts were then reconstituted to 25 pL by dissolution in 80% vol methanol and were stored at 4 °C before analysis.

[0041] Instrumental DI-MS/MS Operating Conditions - DI-MS/MS was performed using an Agilent 7100 capillary electrophoresis (CE) system (Agilent Technologies Inc., Mississauga, ON, Canada), which was used to inject and flush sample solutions into an Agilent 6550 quadrupole-time-of-flight (Q-TOF) mass analyzer. A 75 cm (total length) uncoated fused-silica capillary (75 pm I.D., 360 pm O.D.) from Polymicro Technologies Inc. (Phoenix, AZ) was used for DI after 1 cm of the polyimide outer coating was burnt off both ends of a capillary using a capillary windowmaker (MicroSolv Technologies Inc., Leland, NC, USA). The CE-MS system used an Agilent electrospray ionization (ESI) coaxial sheath liquid interface with an Agilent 1260 Infinity isocratic pump and a 1260 Infinity degasser (Agilent Technologies Inc.) to deliver a sheath liquid consisting of 60:40 methanol-water with 0.1 % vol formic acid at a rate of 1 mL/min. A 1 : 100 splitter was used to reduce the sheath liquid volume to the sprayer to 10 //L/min. For realtime mass correction, reference ions for purine, hexamethoxyphosphazine (HP-0321) and hexakis(2,2,3,3-tetrafluoropropoxy)phosphazine (HP-0921) were spiked into the sheath liquid at 0.02% vol to provide constant mass signals at m/z 121.0509, 322.0481, and 7922.0098, respectively. Optimized parallel reaction monitoring (PRM) transitions for 2-NO-Pyr- cycloadducts of 25OH-D3, 25OH-D2, and d6-25OH-D3 were implemented to lower detection limits as summarized in Table 1.

Table 1

Optimal transitions used for parallel reaction monitoring with a Q-TOF system for 25OH-D metabolites labeled with 2-NO-Pyr.

Precursor Quantifier Fragmeutor Collision Energy

25OH-D metabolites Ion (m/z) Ion (m/z) (AQ (V)

25OH-D3 cycloadduct 509.4 227.1179 120 30

25OH-D2 cycloadduct 521.4 227.1179 120 30 d6-25OH-D3 cycloadduct 515.4 227.1179 120 30

[0042] The Q-TOF system was operated in positive ion mode. Ion source conditions were as follows: the gas temperature at 225 °C, drying gas flow rate at 11 L/min, nebulizer pressure at 60 psi, sheath gas temperature at 125 °C, sheath gas flow rate at 2 L/min, Vcap at 3000 V, nozzle voltage at 2000 V, and fragment at 150 V. During sample introduction, the nebulizer was set to 10 psi, and a sample vial was flushed at 950 mbar for 10 s. The CE instrument was designed to program an alternating series of plasma/serum extracts injection plugs, calibrant solutions and blanks corresponding to about 1.1 //L of total sample volume loaded onto a bare fused-silica capillary. After sample injection, a vial containing 80% vol MeOH was placed at the capillary inlet, the nebulizer pressure was set back to 60 psi, and a pressure of 100 mbar was applied for 2.3 min. This procedure was repeated by programming a high-pressure sample introduction followed by a low-pressure sample infusion using Agilent Mass Hunter software. The quadrupole was set to a narrow mass window (m/z 1.3), and the acquisition rate for both MS and MS/MS was set to 3 spectra/s. Also, the mass range for MS/MS was set from m/z 50 to 600, while the field for fullscan MS spectra was set from m/z 50 to 1700.

[0043] Method Validation, Quality Control, and Inter -method Comparison Study - Calibrants containing 25OH-D3 and 25OH-D2 at 20, 65, 110, 155, 200, and 250 nmol/L in 80% methanol were prepared with 320 nmol/L d6-25OH-D3 as a stable-isotope internal standard from 1.0 mol/L stock solutions in ethanol. Intra-day precision samples were prepared from normal human serum purchased from Sigma Aldrich Inc. Samples were analyzed sequentially in batches of 20 separated by analyzing a single blank solution to check for sample carryover. A fresh solution vial was used for infusion after every 10 sample injections to minimize the sample carryover. Inter-day precision samples were analyzed over three days under conditions to simulate real-world laboratory operating conditions. Each day, a new fused-silica capillary was prepared, the ion source was cleaned, and the instrument was mass tuned.

[0044] Analysis began by running a blank, a 6-point calibration curve, and ten serum extract samples each day. This procedure was repeated five times resulting in the study of 5 blanks, 30 calibrants, and 50 serum samples. Spike and recovery experiments were performed by spiking 25OH-D metabolites at 0, 15, 30, and 45 nmol/L in Sigma serum samples in triplicate. The percent recovery was calculated by subtracting the concentration of the non-spiked samples from that of the spiked samples and then dividing by the known spiked concentration. Stability of 25OH-D cycloadducts was investigated by processing DEQAS serum samples (576-580) and analyzing them after 0, 1, and 2 repeat freeze-thaw cycles when stored at -80 C. External validation was performed to evaluate the accuracy of DI-MS/MS as compared to a commercial chemiluminescence immunoassay system (Qualigen Therapeutics Inc., Carlsbad, CA, USA) by analyzing reference serum samples from NIST and DEQAS. This immunoassay system is currently approved by the FDA and Health Canada, which has been the assay of choice for ICU studies in hospital sites that do not offer rapid 25OH-D screening by their local clinical laboratories. An inter-method comparison of DI-MS/MS with Qualigen immunoassay was also performed on plasma samples analyzed from a cohort of critically ill pediatric patients (n=30).

[0045] Data Processing and Statistical Analysis - All data acquired by DI-MS/MS were analyzed with the Agilent Mass Hunter Workstation Software (Qualitative Analysis, version B.06.00, Agilent Technologies Inc.). Ion chromatograms were extracted in profile mode with a 50 ppm mass window and integrated after smoothing using a Gaussian function (function width: 15, gaussian width: 10). Peak areas were transferred to excel (Microsoft office, Edmond, WA, USA) to calculate relative peak areas (RPAs) using d6-9 25OH-D3 to correct for differences in injection volume, ion suppression, and overall sample workup. All chromatograms were depicted using the Igor Pro 5.0 software (Wavemetric Inc., Lake Oswego, OR, USA). Analysis of external calibration data, calculation of figures of merit, and generation of control charts were performed using Microsoft Excel. Box plots, Passing-Bablok regressions, Bland- Altman % difference plots, and empirical cumulative distribution function plots were generated in R version 4.0.3. Mountain % difference plots were generated using MedCalc (MedCalc Software, Ostend, Belgium).

Results and Discussion

[0046] A DI-MS/MS Protocol for Rapid Screening of Vitamin D Nutritional Status - 2-NO-

Pyr was first synthesized via a two-step procedure that 1H-NMR and characterized MS/MS (Figure 1). Ion suppression effects in ESI can be minimized in DI-MS/MS when using a stable-isotope internal standard(s) with optimal sample pretreatment procedures. In the present case, a modified liquid-phase extraction protocol using a 50 pL aliquot of serum or plasma was developed to significantly reduce ion suppression by 7-8-fold due to excess reagent and reaction by-products (Figure 2) following click derivatization when incorporating an alkaline aqueous back extraction step in hexane. Chemical labelling introduces a cationic pyridinium moiety, which increased solute ionization efficiency by over 10-fold under positive ion mode detection relative to unlabeled 25OH-D (Figure 3) with quantitative 25OH-D cycloadduct formation (> 95%) via a Diels-Alder reaction (10 mM 2-NO-Pyr, 60 C at 30 min). Figure 4A highlights that CE was configured to perform DI by programming a repeated sequence comprising a long hydrodynamic sample volume injection (~ 1.1 pL on-capillary) followed by a low pressure (100 mbar or 10 kPa) solvent flush with 80% vol methanol when coupled to a coaxial sheath liquid interface. The total duty cycle was 3.3 min per sample, including delay times for automatic changing vials on the sample carousel, where 25OH-D3 and 25OH-D2 cycloadducts are monitored via PRM (Table 2) as shown in a representative MS/MS spectrum (Figure 4B). Moreover, data processing after Gaussian smoothing generated average peaks (13 data points collected over a base peak width of ~ 1 min; Figure 5) with data normalized to d6-25OH-D3 as an internal standard to correct variations in injection volume, ion suppression, and sample handling. Table 2.

Summary of key figures of merit for the determination of 25OH-D in plasma and serum extracts by DI-MS MS with 2-NO-Pyr click derivatization.

Figures of merit 25OH-D3 25OH-D2

Mean recovery (range)’ 103% (92 to 112%) 101% (93 to 113%)

Precision (intraday: interday) ^b 10.9 %: 10.5 % X A: X A

Lineality (slope: R- 0.0037; 0.992 0.0037: 0.996

LOQ (SZV = 10) 3.9 nmol L (1.7 pg) 3.6 nmol L (1.6 pg)

LOD (S/N = 3) 1.2 nmol L (0.52 pg) 1.1 nmol L (0.50 pg)

% Detects in reference blood samples (n=18) ^d 100% 21%

% Detects in pediatric plasma samples (n=30) ^e 100% 43%

[0047] Method Validation of DI-MS/MS for Reliable 25OH-D Determination - Next, method validation of the DI-MS/MS assay was conducted, including a repeat analysis of Sigma serum extracts continuously over a 24 h period generated good reproducibility for 25OH-D3 quantification (mean CV = 10.9%, n=412) without evidence of sample carry-over for blank samples (n=20) analyzed intermittently (Figure 4C, D). Overall, excellent linearity (R ² >0.999) was achieved for 6-point calibration curves measured in triplicate over a wide dynamic range (20- 250 nmol/L, mean CV = 8.6%) for both 25OH-D3 and 25OH-D2 (Figure 4E, F) with quantification limits (S/N ~ 10) and detection limits (S/N ~ 3) of about one nmol/L and four nmol/L, respectively (Table 2). Moreover, spike-recovery studies (at 15, 30 and 45 nmol/L in triplicate) confirmed acceptable accuracy for serum 25OH-D3 and 25OH-D2 determination by DI-MS/MS with a mean recovery of (102 ± 12) % ranging from 92 to 113% (Figure 4G; Table 2). Also, intermediate precision when measuring serum extracts over three days by DI-MS/MS demonstrated adequate reproducibility (mean CV = 10.5%, n=150; Figure 6), whereas analysis of 25OH-D from plasma extracts after two repeat freeze-thaw cycles confirmed good chemical stability without degradation of labelled vitamin D cycloadducts (Figure 7).

[0048] Inter-method Comparison for Serum 25OH-D Determination in Reference Samples

- An external validation of DI-MS/MS for 25OH-D determination by analyzing blood samples from two quality assessment providers (DEQAS, n=15; NIST, n=3) in duplicate, which were also measured by LC-MS/MS as a reference method and a commercial chemiluminescence immunoassay system. Figure 8A, B highlights good mutual agreement in 25OH-D quantification by DI-MS/MS compared to LC-MS/MS with acceptable linearity (slope = 1.17) and a mean bias of 7.8% (n=18). This positive bias was mainly attributed to co-elution of 3-epi-25OH-D3 to total 25OH-D as measured by DI-MS/MS, which was confirmed after adjusting the 3-epimer that reduced the mean bias relative to LC-MS/MS to only 1.5% (Figure 9). Automated immunoassays with bias < 10% have been applied safely in clinical practice. However, an inter-method comparison of immunoassay and LC-MS/MS for the same reference blood samples confirmed a more considerable mean bias of 12.9% (Figure 10), reflecting lower method selectivity. There was an appreciable positive bias (42%) for immunoassay from 3-epi-25OH-D3 enriched in the NIST L4 sample with nearly equimolar concentrations to 25OH-D3 (Figure 11). Also, the immunoassay was prone to batch effects when using five different reagent lots (Figure 12) with more considerable variability for QC samples (mean CV = 18%, n=52) when analyzed over ten months.

[0049] Impact of Method Bias When Screening for Vitamin D Deficient, Critically III Children - Figure 8C depicts a Mountain plot overlay which confirms that DI-MS/MS (-4.1%) generated a lower median bias than immunoassay (-9.6%) when both were compared to LC- MS/MS for reference serum samples were analyzed. The greater extent of the bias for the immunoassay has important consequences when classifying vitamin D deficient (< 50 nmol/L) children who may clinically benefit from vitamin D supplementation yet may not be recruited if relying on immunoassay results. Figure 8D depicts empirical cumulative frequency distribution plots for plasma 25OH-D concentrations from pediatric patients (n=30) for their potential recruitment in a randomized control trial using immunoassay or DI-MS/MS as the primary screening method. As expected, there was a lower mean 25OHD concentration reported for all potential participants (-5.0 nmol/L) for DI-MS/MS relative to immunoassay (59.7 Vs. 64.7 nmol/L), which corresponded to a more significant fraction of participants being classified as vitamin D deficient (13 of 30 or 43% Vs. 5 of 30 or 16.7%). Plasma 25OH-D concentration ranges were also more comprehensive in this cohort when measured by DI-MS/MS than immunoassay due to its higher limit of quantification (LOQ ~ 32 nmol/L), which is also prone to matrix effects in blood samples. As a result, precise and accurate screening for 25OH-D determination is critical to authenticate vitamin D deficient children likely responsive to therapeutic vitamin D interventions.

[0050] Thus, in this embodiment of the invention, the DI-MS/MS method was used to determine plasma 25-OH-D concentrations in a cohort of critically ill children (n=30), which was validated in certified reference serum samples (n=l 8) independently measured by a FDA-approved commercial immunoassay and a validated LC-MS/MS protocol. In this embodiment, an optimized liquid-phase extraction protocol was developed to minimize ion suppression when directly infusing serum or plasma extracts via a capillary electrophoresis system used in DI for the quantitative determination of 25OH-D. This embodiment achieved greater throughput and less bias than a commercial chemiluminescence immunoassay, which is also prone to batch effects. In this embodiment, there was reduced immunoassay misclassification of vitamin D deficiency in a cohort of hospitalized children in intensive care. In this embodiment, DI-MS/MS offered a reliable alternative to liquid chromatography-tandem mass spectrometry (LC-MS/MS) for routine screening of vitamin D status.