Methods and systems for spectrophotometric detection of γ-tocopherol via nitrogen oxide-induced colorimetric reaction products

Abstract

A method for spectrophotometric detection of γ-tocopherol in a sample, comprising mixing the sample with a reagent composition in a solvent to form a mixture; incubating the mixture; measuring absorbance of the mixture; and correlating the measured absorbance to γ-tocopherol concentration in the sample.

Description

Attorney Docket No. : 3229-6 PCTMETHODS AND SYSTEMS FOR SPECTROPHOTOMETRIC DETECTION OF y- TOCOPHEROL VIA NITROGEN OXIDE-INDUCED COLORIMETRIC REACTION PRODUCTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U. S. Provisional Patent Application No. 63 / 689,622 filed on August 30, 2024, the entire contents of which are incorporated by reference herein.TECHNICAL FIELD

[0002] The subject matter of the present disclosure relates generally to methods and systems for spectrophotometric detection of y-tocopherol via nitrogen oxide-induced colorimetric reaction products and, in particular, to methods and systems for quantitatively determining y-tocopherol levels in biological fluids, foods, oils, and other materials.BACKGROUND

[0003] Current analytical methods intended for the determination of y-tocopherol in biological fluids, foods, oils, and other matrices are frequently inadequate in their performance, accessibility, and scalability. Conventional assays often rely on high performance liquid chromatography or gas chromatography, frequently coupled with mass spectrometry, which require specialized instrumentation, costly consumables, and highly trained personnel. While such chromatographic methods can achieve high sensitivity, they are not well suited for widespread clinical, industrial, or field applications. Existing methodologies present additional limitations including lengthy sample preparation steps such as saponification or multi-stage solvent extraction, susceptibility to interference from coextracted lipids and other matrix constituents, and stringent requirements for instrumentAttorney Docket No. : 3229-6 PCT calibration and maintenance. These burdens can introduce analytical variability, extend turnaround times, and significantly increase operational costs. As a result, y-tocopherol measurement is not routinely incorporated into clinical assessments, nutritional screening, or quality control workflows despite its recognized value as a biomarker of oxidative stress, inflammation, and dietary intake. Furthermore, conventional laboratory-based methods are poorly suited to decentralized testing environments, since they depend on infrastructure that is unavailable in many clinical, community, or field settings. The inability to perform rapid, low-cost, and reliable measurements restricts the integration of y-tocopherol screening into routine health monitoring, quality control of edible oils, or large-scale epidemiological studies where timely assessment of antioxidant status could inform medical, nutritional, or processing decisions.

[0004] Accordingly, there remains a need for methods and systems for spectrophotometric detection of y-tocopherol that exploit nitrogen oxide-induced formation of distinct colorimetric reaction products. In particular, there is a need for methods and systems that allow for the quantitative determination of y-tocopherol levels across a wide range of sample types using accessible equipment such as standard spectrophotometers or plate readers, minimal technical training, and cost-effective reagents.SUMMARY

[0005] In accordance with aspects of the disclosure, a method for spectrophotometric detection of y-tocopherol in a sample includes: mixing the sample with a reagent composition in a solvent to form a mixture; incubating the mixture; measuring absorbance of the mixture; and correlating the measured absorbance to y-tocopherol concentration in the sample.Attorney Docket No. : 3229-6 PCT

[0006] In an aspect of the present disclosure, the method may further include incubating the mixture at about 45 °C for about 8 to about 10 hours.

[0007] In an aspect of the present disclosure, the method may further include measuring the absorbance of the mixture at a wavelength in the range of about 420 nm to about 450 nm.

[0008] In an aspect of the present disclosure, the reagent composition may include a nitrogen oxide-producing chemical.

[0009] In an aspect of the present disclosure, the nitrogen oxide-producing chemical may include morpholinosydnonimine hydrochloride.

[0010] In an aspect of the present disclosure, the method may further include dissolving the morpholinosydnonimine hydrochloride in methanol such that the methanol constitutes no more than about 10% by volume of the mixture.

[0011] In an aspect of the present disclosure, the solvent may include dichloromethane.

[0012] In an aspect of the present disclosure, the method may further include incubating the mixture at about 45 °C for about 8 to about 14 hours in the absence of light.

[0013] In an aspect of the present disclosure, the method may further include forming a chromophoric reaction product including at least one of: 2,7,8-trimethyl-2-(4,8,12- trimethyltridecyl)-5,6-chromaquinone (tocored); or 2,7,8-trimethyl-2-(4,8,12- trimethyltridecyl)-5-nitro-6-chromanol (tocoyellow).

[0014] In an aspect of the present disclosure, the method may further include generating a calibration curve by treating a series of concentrations of authentic y-tocopherol with the nitrogen oxide-producing chemical.

[0015] In accordance with aspects of the disclosure, the method may further include, prior to the mixing step, combining the sample with ethanol to form a pre-extraction mixture.Attorney Docket No. : 3229-6 PCT

[0016] In an aspect of the present disclosure, the method may further include extracting the pre-extraction mixture with hexane to form a hexane layer.

[0017] In an aspect of the present disclosure, the method may further include subjecting the hexane layer to solid phase extraction on a cartridge.

[0018] In an aspect of the present disclosure, the method may further include eluting y- tocopherol from the cartridge with a mixture of hexane and a solvent to obtain an eluate.

[0019] In an aspect of the present disclosure, the method may further include evaporating the eluate to dryness and redissolving a residue resulting from the evaporating in the solvent.

[0020] In an aspect of the present disclosure, the sample may include a biological fluid, a food product, a dietary supplement, or a plant extract.

[0021] In an aspect of the present disclosure, the cartridge may be a silica gel cartridge.

[0022] In an aspect of the present disclosure, the method may further include incubating the mixture at about 45°C for about 10 to about 14 hours in the absence of light.

[0023] In accordance with aspects of the disclosure, a method for spectrophotometric detection of y-tocophcrol in a biological fluid includes: mixing the biological fluid with ethanol in a vessel using a mixing device to form a mixture; extracting the mixture with hexane to form a hexane layer; subjecting the hexane layer to solid-phase extraction on a cartridge; eluting v- tocopherol from the cartridge with a mixture of hexane and a solvent to derive an eluate; evaporating the eluate to dryness; redissolving formed residue in the solvent; mixing the redissolved formed residue with a reagent composition using a mixing device to form a reaction mixture; incubating the reaction mixture; measuring absorbance of the reaction mixture using a spectrophotometer; and correlating the measured absorbance to y-Attorney Docket No. : 3229-6 PCT tocopherol concentration in the biological fluid using a calibration curve generated from authentic y-tocophcrol standards.

[0024] In accordance with aspects of the disclosure, a system for spectrophotometric detection of y-tocopherol in a sample includes: a solid-phase extraction cartridge including silica gel configured to isolate y-tocopherol from the sample; a vessel configured for incubation of a mixture at about 45 °C for about 10 to about 1 hours in the absence of light to produce tocored and tocoyellow, the mixture formed by a reagent composition including morpholinosydnonimine hydrochloride dissolved in methanol and combined with dichloromethane; and a spectrophotometric device configured to measure absorbance of the mixture at about 435 nm and determine y-tocopherol concentration using a calibration curve prepared from authentic y-tocopherol standards. The solid-phase extraction cartridge may be configured to isolate y-tocopherol from the sample and deliver the isolated y-tocopherol for incubation in the vessel.

[0025] Further details and aspects of exemplary embodiments of the present disclosure are described in more detail below with reference to the appended figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings of which:

[0027] FIG. 1 is an illustration of an exemplary method for spectrophotometric detection of y-tocopherol in a sample, in accordance with aspects of the present disclosure;Attorney Docket No. : 3229-6 PCT

[0028] FIG. 2 is an illustration of an exemplary method for spectrophotometric detection of y-tocopherol in a biological fluid, in accordance with aspects of the present disclosure;

[0029] FIG. 3 is an illustration of an exemplary system for spectrophotometric detection of y-tocopherol in a sample, in accordance with aspects of the present disclosure; and

[0030] FIG. 4 is a block diagram of a controller configured for use with the system of FIG.3, in accordance with aspects of the present disclosure.DETAILED DESCRIPTION

[0031] The present disclosure relates generally to methods and systems for spectrophotometric detection of y-tocopherol via nitrogen oxide induced colorimetric chromophoric reaction products and in particular to methods and systems that allow for the quantitative determination of y-tocopherol levels across a wide range of sample types using accessible equipment such as standard spectrophotometers or plate readers, minimal technical training, and cost-effective reagents.

[0032] Although the present disclosure will be described in terms of specific examples, it will be readily apparent to those skilled in this art that various modifications, rearrangements, and substitutions may be made without departing from the spirit of the present disclosure.

[0033] For the purpose of promoting an understanding of the principles of the present disclosure, reference will now be made to exemplary embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the present disclosure is thereby intended. Any alterations and further modifications of the novel features illustrated herein, and any additional applications of the principles of the present disclosure as illustrated herein, which would occurAttorney Docket No. : 3229-6 PCT to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the present disclosure.

[0034] Referring to FIG. 1, a method 100 for spectrophotometric detection of y-tocopherol in a sample 11 is shown. At step 102, the method 100 includes preparing and mixing a sample 11 with a reagent composition 12 in a solvent 13 in a vessel 18 using a mixing device 10 to form a mixture 14. The sample 11 may be obtained from a wide range of sources, including but not limited to biological fluids such as plasma, serum, urine, saliva, cerebrospinal fluid, or whole blood. In certain embodiments, the sample 11 may include additional clinical or biological matrices such as milk, synovial fluid, amniotic fluid, sweat, or tissue homogenates derived from liver, adipose, or muscle. The method 100 may also be applied to animal-derived fluids or experimental samples in preclinical studies, extending its applicability beyond human testing. Food-derived materials are also suitable and may include vegetable oils, nut oils, seed oils, grains, dietary supplements, fortified beverages, edible fats, or processed food extracts. In research, regulatory, or quality control contexts, the sample 11 may be prepared as purified tocopherol standards dissolved in an appropriate solvent 13 system for calibration and validation.

[0035] The sample 11 may also be subjected to pretreatment steps prior to combination with the reagent composition 12. Such pretreatment may include liquid-liquid extraction with hexane or dichloromethane, protein precipitation with alcohols such as ethanol, solid-phase extraction on silica cartridges, or partitioning against nonpolar solvents to enrich y-tocopherol relative to other matrix constituents. Additional treatments such as filtration, centrifugation, or dialysis may be employed to clarify the sample 11 and remove interfering proteins, lipids, or pigments. In some embodiments, antioxidants or stabilizers may be added to the sample 11Attorney Docket No. : 3229-6 PCT to preserve y-tocopherol during preparation. Pretreatment reduces complexity and improves the accuracy and reproducibility of subsequent spectrophotometric analysis while ensuring compatibility with the solvent 13 and the nitrogen oxide-based derivatization chemistry.

[0036] Mixing step 102 may be performed using the mixing device 10, which may include a vortex mixer, an overhead stirrer, a magnetic stirrer, an orbital shaker, a rotary tumbler, a sonicator, or any other agitation apparatus configured to achieve homogeneous distribution of the reagent composition 12 within the sample 11. Where the sample 11 includes biphasic mixtures of aqueous and organic solvents, the mixing device 10 may be selected to promote phase contact and efficient extraction of y-tocopherol, with vortexing, sonication, or orbital shaking being particularly effective. In high-throughput embodiments, robotic mixing stations, automated pipetting systems, or microfluidic mixing devices may be employed, allowing for simultaneous processing of large numbers of samples in multiwell plates or cartridge-based arrays. In certain embodiments, the mixing device 10 may be integrated with temperature control or sealed reaction vessels 18 to minimize solvent 13 loss and ensure reproducibility, particularly when volatile solvents 13 such as dichloromethane are used. In low-resource or field-deployable settings, manual agitation such as inversion, pipette aspiration and dispensing, or mixing by capillary action within microfluidic channels may suffice.

[0037] The reagent composition 12 may include a nitrogen oxide-producing chemical, such as morpholinosydnonimine hydrochloride, which under reaction conditions decomposes to release nitric oxide and nitrogen dioxide. These reactive species interact with y-tocopherol to induce oxidation and nitration, producing distinct chromophoric derivatives detectable by spectrophotometry. Other nitrogen oxide-producing chemicals may also be employed,Attorney Docket No. : 3229-6 PCT including nitrosothiols such as S-nitrosoglutathione, peroxynitrite-generating systems, sodium nitroprusside, diazeniumdiolate salts, or related donors of nitrosating or nitrating species, provided that they selectively transform y-tocopherol into detectable reaction products 17. In some embodiments, inorganic nitrite salts in acidic conditions or nitrosyl halides may serve as alternative sources of nitrogen oxide reactivity.10038] The nitrogen oxide-producing chemical of the reagent composition 12 may be supplied as a powder, a pre-dosed ampule, or a stabilized stock solution. It may be dissolved in a co-solvent such as methanol, ethanol, isopropanol, acetonitrile, or other polar solvents 13 compatible with the reaction system. In certain embodiments, methanol is used and controlled such that it constitutes no more than about 10% by volume of the mixture 14. This limitation minimizes solvent 13 interference with subsequent spectrophotometric analysis while maintaining solubility of the donor compound.

[0039] The solvent 13 may include di chloromethane, which provides a low-background medium for spectrophotometric detection, or another organic solvent 13 of similar polarity and volatility such as chloroform, tetrahydrofuran, ethyl acetate, or toluene. The choice of solvent 13 may be dependent upon considerations of volatility, compatibility with the nitrogen oxide donor, and stability of chromophoric reaction products 17. In some embodiments, binary or ternary mixtures of solvents 13 may be employed to optimize solubility of both the reagent composition 12 and the sample 11. Additives such as buffers, chelators, or stabilizers may also be included to control pH, limit competing oxidation, or improve reproducibility.

[0040] The vessel 18 may be a sealed vial, a cuvette, a test tube, a microplate well, or a microfluidic chamber, depending on throughput, sample 11 volume, and analytical design. In certain embodiments, the vessel 18 may include a standard glass or plastic vial with a crimpedAttorney Docket No. : 3229-6 PCT or screw-cap closure, allowing convenient sealing for protection from evaporation and light. In spectrophotometric cuvette-based assays, the vessel 18 may include quartz or optical-grade plastic cuvettes with defined path lengths (e.g., 1 cm, 5 mm, or 2 mm), promoting direct transfer from incubation to measurement without additional handling. In laboratory-scale testing, the vessel 18 may be a disposable polypropylene or borosilicate test tube capable of withstanding organic solvents 13 and incubation temperatures. For high-throughput or automated workflows, the vessel 18 may be a multiwell plate, such as a 96- well or 384- well plate, constructed from solvent-resistant and optically transparent materials to facilitate simultaneous processing and detection of many samples 11 in parallel. In some embodiments, the vessel 18 may include microfluidic chips or cartridge-based chambers incorporating microchannels, reaction reservoirs, and integrated optical windows, allowing miniaturization and point-of-care deployment of the assay.

[0041] Additional configurations of the vessel 18 may include amber-colored or foilwrapped containers designed to block ambient light, thereby preserving light-sensitive chromophoric reaction products 17. In certain embodiments, the vessel 18 may also incorporate inert liners, septa, or coatings to minimize adsorption of y-tocophcrol or reaction intermediates to vessel 18 walls. Where volatile solvents 13 such as dichloromethane are employed, the vessel 18 may include gas-tight seals or be maintained under an inert gas atmosphere such as nitrogen or argon. The vessel 18 may also be provided in disposable, presterilized form for clinical use or in reusable, autoclavable formats for laboratory research.

[0042] At step 104, the method 100 includes incubating the mixture 14 under controlled conditions within an incubator 15. Incubation is conducted at a temperature of about 45 °C,Attorney Docket No. : 3229-6 PCT although temperatures ranging between about 30°C and about 60°C may be employed depending on the desired reaction kinetics. A typical incubation period is about 8 to 10 hours, although incubation times between about 2 hours and about 24 hours are contemplated, particularly when alternative nitrogen oxide donors are employed. The incubation may be performed in the absence of light to avoid photodegradation or interference from photoinduced reactions, although amber or opaque vessels may also be used to minimize light exposure. The incubator 15 may include water baths, dry block heaters, incubator cabinets, temperature-controlled reaction blocks, microplate heating units, or other devices configured to maintain uniform heating across the vessel 18. In embodiments, heating may be supplemented or replaced by microwave irradiation, ultrasonic agitation, or infrared heating. During incubation, the y-tocopherol present in the sample 11 undergoes chemical transformation through interaction with reactive nitrogen oxide species derived from the reagent composition 12, producing distinct reaction products 17 that may be chromophoric in nature.

[0043] Reaction products 17 may include tocored and tocoyellow as primary species. Tocored is chemically identified as 2,7,8-trimethyl-2-(4,8,12-trimethyltridecyl)-5,6- chromaquinone, a quinone derivative of y-tocopherol formed through oxidation of the chromanol ring. Tocoyellow is chemically identified as 2,7,8-trimethyl-2-(4,8,12- trimethyltridecyl)-5-nitro-6-chromanol, a nitrochromanol derivative of y-tocopherol resulting from nitration of the chromanol moiety. These compounds absorb light strongly in the visible spectrum, particularly in the range of about 420 nanometers to about 450 nanometers, with typical absorbance maxima centered near 435 nanometers when dichloromethane is used as the solvent 13.Attorney Docket No. : 3229-6 PCT

[0044] The formation of these reaction products 17 provides a distinct, measurable signal that directly correlates with the amount of y-tocopherol originally present in the sample 11. The relative proportions of tocored and tocoyellow may vary depending on reaction conditions, including incubation time, temperature, exclusion of light, choice of solvent 13, and the specific nitrogen oxide donor employed. For example, methanol content above 10% by volume may alter product yields, while alternative solvents 13 such as chloroform or ethyl acetate may favor different chromophoric ratios.

[0045] In addition to tocored and tocoyellow, other minor derivatives may also form, including partially oxidized, dimerized, or rearranged species of y-tocopherol. While these products are less prominent, their presence can provide supplementary absorbance signals and extend the dynamic range of the assay. In some embodiments, analysis of the combined absorbance profile across 420-450 nanometers allows for quantitation even when product ratios shift under varying conditions. The reproducible generation of strongly absorbing visible chromophores ensures that method 100 provides reliable detection of y-tocopherol without reliance on high-performance chromatographic separation.

[0046] At step 106, the method 100 includes measuring absorbance of the mixture 14 using the spectrophotometer 16 or an equivalent detection device. The measurement is performed at a wavelength in the range of about 420 nanometers to about 450 nanometers, and preferably at about 435 nanometers when dichloromethane is used as the solvent 13, which provides a stable absorbance maximum for tocored and tocoyellow. In some embodiments, full spectral scans across the visible range may be performed, allowing confirmation of product identity and background correction in addition to single-wavelength monitoring.Attorney Docket No. : 3229-6 PCT

[0047] The spectrophotometer 16 may be a bench-top cuvette reader, a microplate spectrophotometer, a portable optical photometer, or an integrated photometric detection unit configured to detect absorbance of visible light. Alternative formats may include fiber-optic probes coupled to a detection unit for in situ monitoring, integrated incubator-detector modules that combine heating and measurement in a single device, or multi-channel instruments capable of reading dozens to hundreds of samples in parallel. In some embodiments, high-throughput analysis may be performed using a multiwell plate format and a plate reader configured with optical filters or monochromators suitable for the 420-450 nm range.

[0048] For point-of-care or field-deployable applications, the spectrophotometer 16 may be embodied as a miniaturized handheld device, a smartphone-coupled photometer, or a disposable strip-based optical reader. Such configurations allow rapid, low-cost measurement outside of laboratory settings. The mixture 14 may be transferred to an optical cuvette, a microplate well, or retained in the vessel 18 in which incubation occurred, depending on the requirements of the spectrophotometer 16. In certain embodiments, sealed or solvent -resistant cuvettes may be preferred to minimize evaporation of volatile solvents 13 such as dichloromethane .

[0049] The absorbance signal may be recorded digitally using instrument software, stored in laboratory information management systems, or processed by automated calibration algorithms to directly output y-tocopherol concentration. In some embodiments, results may be wirelessly transmitted to remote databases, cloud-based storage, or clinical decision support systems. Manual readout using conventional instrument displays or spectrophotometer strip readers may also be employed in low-resource environments. TheAttorney Docket No. : 3229-6 PCT recorded absorbance represents the intensity of chromophore formation under the specified reaction conditions and provides a reproducible optical signature that directly correlates to y- tocopherol content.

[0050] At step 108, the method 100 further includes correlating the measured absorbance to the y-tocopherol concentration in the original sample 11. This correlation is performed using a calibration curve that is generated from authentic y-tocopherol standards subjected to the same chemical and physical conditions as the sample 11. To establish the calibration curve, a series of known concentrations of authentic y-tocopherol are treated with the reagent composition 12 in the solvent 13, incubated in the incubator 15 under comparable conditions, and analyzed with the spectrophotometer 16. The absorbance values measured for each concentration are plotted to generate a calibration profile, which may be linear or non-linear depending on concentration range, solvent 13 background, and optical response of the detection system.

[0051] In some embodiments, statistical tools such as multiple regression models, polynomial fitting, or computational algorithms may be applied to refine the correlation and extend accuracy across a wider concentration range. Calibration curves may be constructed using standards prepared directly in solvent 13 or in matrix- matched materials such as stripped plasma, serum, or refined oil, thereby accounting for potential background interference. Internal standards such as 5-tocopherol, a-tocopherol, or other structurally related analogs may be included to normalize results, improve reproducibility, and control for extraction variability, solvent 13 loss, or incubation inconsistencies.

[0052] The correlation step may be performed manually by plotting values and interpolating results, or automatically through instrument software that integrates calibrationAttorney Docket No. : 3229-6 PCT data into routine analysis. Tn some embodiments, the calibration curve may be periodically refreshed or stored digitally to maintain consistency across multiple analytical runs. The absorbance of the sample 11 is then compared to the calibration curve to provide a quantitative determination of y-tocopherol concentration that is reproducible across biological fluids, food materials, and research samples 11.

[0053] The method 100 provides a robust framework for detecting and quantifying y- tocopherol in a wide variety of sample 11 matrices. By combining controlled chemical derivatization with incubation under reproducible thermal and light-controlled conditions, followed by standardized spectrophotometric detection and calibration, the method 100 achieves accurate and reproducible quantitation of y-tocopherol. The breadth of potential solvents 13, reagent compositions 12, mixing devices 10, incubation conditions within the incubator 15, and spectrophotometers 16 supports wide applicability across laboratory, clinical, research, and industrial contexts.

[0054] In addition, the ability to generate distinct chromophoric reaction products 17 such as tocored and tocoyellow ensures sensitive detection and provides an unambiguous spectral signature for y-tocopherol, reducing interference from other tocopherol isomers or sample 11 constituents. Alternative nitrogen oxide donors, solvent 13 systems, and incubation formats may be selected to adapt the method 100 for different resource levels or throughput requirements. This adaptability allows the method 100 to be applied to small-scale laboratory assays for mechanistic studies, high-throughput screening of population-based samples in epidemiological research, field-based diagnostics in decentralized or point-of-care environments, or industrial quality control of nutritional supplements, fortified foods, and edible oils.Attorney Docket No. : 3229-6 PCT

[0055] In some embodiments, the method 100 may be integrated into automated platforms that combine extraction, derivatization, incubation, and detection into a single streamlined process. In other embodiments, simplified kits containing pre-dosed reagents 12 and portable spectrophotometers 16 may extend utility into community health programs or agricultural monitoring. The stability and reproducibility of the chromophoric signal across diverse solvents 13 and incubation regimes underscores the reliability of the method 100, supporting its use as a practical, cost-effective, and broadly accessible tool for y-tocopherol measurement.

[0056] Referring now to FIGS. 2 and 3, an exemplary method 200 for spectrophotometric detection of y-tocopherol in a biological fluid 21 and a system 300 for spectrophotometric detection of y-tocopherol in a sample 11 (e.g., a biological fluid 21) are shown. The biological fluid 21 may include plasma, serum, urine, saliva, milk, synovial fluid, amniotic fluid, or other clinically relevant matrices. In general, method 200 extends the principles described for method 100 by incorporating extraction, isolation, and cleanup operations prior to chemical derivatization, thereby allowing accurate detection of y-tocopherol even in samples 11 that contain lipids, proteins, or other potential interferences.

[0057] At step 202, the method 200 includes mixing a biological fluid 21 with a volume of ethanol 22 in vessel 18 using mixing device 10 to form a mixture 23. The biological fluid 21 may include plasma or serum, and in certain embodiments may also include other blood- derived fluids such as whole blood, platelet-rich plasma, leukocyte-depleted plasma, or lysed blood fractions. Additional embodiments may encompass biological fluids 21 such as saliva, cerebrospinal fluid, synovial fluid, amniotic fluid, urine, or milk, all of which can contain detectable amounts of y-tocopherol. Tissue homogenates or extracts prepared by solvent maceration or enzymatic digestion may also serve as the biological fluid 21. The ethanol 22Attorney Docket No. : 3229-6 PCT functions to precipitate proteins and denature enzymatic activities, while simultaneously creating a clarified environment where lipophilic compounds like y-tocopherol are more readily accessible for extraction. Ethanol 22 may be used alone or in combination with other miscible solvents 28 such as methanol, isopropanol, or acetonitrile. The relative proportion of ethanol 22 to the biological fluid 21 may range widely, from about 0.5:1 to about 10:1 by volume, depending on the protein content of the biological fluid 21 and the desired level of precipitation.

[0058] The mixing device 10 at step 202 may employ the same mixers and mixing approaches described for step 102 of method 100, thereby ensuring uniform interaction between the biological fluid 21 and ethanol 22 regardless of the chosen mixing approach.

[0059] At step 204, the method 200 includes extracting the mixture 23 with a volume of hexane 25 to form a hexane layer 26. The hexane 25 serves as a non-polar extraction solvent that partitions y-tocopherol and other hydrophobic molecules away from the ethanol-rich phase, thereby concentrating the analyte into a cleaner fraction for subsequent processing. Alternative solvents to hexane 25 may include isooctane, petroleum ether, heptane, cyclohexane, or other non-polar solvents of comparable volatility and selectivity. In some embodiments, combinations of hexane 25 with low levels of more polar modifiers such as ethyl acetate, diethyl ether, or dichloromethane may be introduced to fine-tune recovery and reduce co-extraction of interfering compounds.

[0060] The extraction process may be carried out by a variety of mixing approaches to maximize phase contact and partition efficiency. These may include vigorous vortexing, manual shaking, orbital mixing, rotary tumbling, or ultrasonic agitation. In high-volume or automated contexts, robotic mixing stations may perform programmed agitation cycles toAttorney Docket No. : 3229-6 PCT ensure reproducibility. After mixing, phase separation may be achieved by gravity settling, centrifugation at moderate speed, or phase separation membranes designed to isolate the nonpolar solvent fraction. In certain embodiments, cold temperature storage may be applied briefly to accelerate partitioning and improve clarity of the separated hexane layer 26.

[0061] The separated hexane layer 26 may be recovered by decanting, pipetting, or withdrawal using automated liquid handling systems. To maximize y-tocopherol recovery, multiple sequential extractions may be performed, with the resulting hexane layers 26 pooled together before proceeding to solid phase cleanup. Such pooled extracts may be combined under nitrogen to minimize oxidation or evaporative loss. In some embodiments, antioxidants such as butylated hydroxytoluene may be added at trace levels to stabilize y-tocopherol during the extraction process.

[0062] At step 206, the method 200 includes subjecting the hexane layer 26 to solid phase extraction on a cartridge 27. The cartridge 27 may contain silica gel 40 as the primary sorbent material, which retains phospholipids, triglycerides, and other interfering lipophilic components while allowing y-tocopherol to be selectively eluted. Alternative sorbent materials may include alumina for adsorption of acidic and polar contaminants, C18 or C8 bonded silica for hydrophobic interactions, ion exchange resins for removal of charged impurities, polymeric reversed phase resins for improved chemical stability, or mixed mode sorbents that combine hydrophobic and ionic retention mechanisms to enhance selectivity.

[0063] The cartridge 27 may be provided in different physical formats depending on throughput and application, such as, standard single-use column cartridges 27 suitable for manual operation, 96-well micro solid phase extraction plates designed for high throughput screening, or integrated modules incorporated into automated solid phase extractionAttorney Docket No. : 3229-6 PCT manifolds and robotic sample preparation stations. Pre-conditioning of the cartridge 27 with solvents such as hexane 25, dichloromethane, or other low polarity solvents may be employed to activate the sorbent surface and improve reproducibility of y-tocopherol recovery. In certain embodiments, the cartridge 27 may also be washed with one or more intermediate solvents, such as hexane 25 with a small percentage of ethyl acetate, to remove weakly retained impurities while minimizing loss of y-tocopherol.

[0064] Flow through the cartridge 27 may be accomplished by gravity, vacuum-assisted manifolds, or positive pressure systems depending on laboratory infrastructure and desired speed of processing. The use of disposable cartridges 27 can reduce cross-contamination, while reusable solid phase extraction (SPE) devices may be employed in industrial settings where high- volume processing and solvent recycling are priorities. This solid phase extraction step provides an important cleanup stage, concentrating y-tocopherol and improving downstream accuracy of spectrophotometric analysis.

[0065] At step 208, the method 200 includes eluting y-tocopherol from the cartridge 27 using a mixture of hexane 25 and a solvent 28. The solvent 28 may include dichloromethane, which is particularly effective in solubilizing y-tocopherol and breaking its interactions with the sorbent surface of the cartridge 27. Other solvents 28 may also be used, including chloroform, acetone, toluene, tetrahydrofuran, or ethyl acetate, each of which provides a different balance of polarity and elution strength. The ratio of hexane 25 to solvent 28 may vary across a broad range, from about 10% hexane 25 to 90% solvent 28, up to about 90% hexane 25 to 10% solvent 28. A typical ratio of about 1 to 1 (1:1) provides effective recovery of y-tocopherol while minimizing co-elution of contaminants. Solvent 13 and solvent 28 areAttorney Docket No. : 3229-6 PCT used interchangeably to denote the solvent employed in the respective methods (100, 200), and both reference numbers represent the same functional element.

[0066] In certain embodiments, a stepwise gradient of hexane 25 and solvent 28 may be applied to gradually displace impurities and then release y-tocophcrol in a more purified fraction. Sequential washes with lower polarity mixtures followed by higher polarity mixtures may further refine recovery and selectivity. Elution may be carried out manually by passing solvents 28 through the cartridge 27 with syringes, pipettes, or gravity flow. Alternatively, elution may be performed automatically with vacuum manifolds, positive pressure devices, or robotic liquid handling systems designed for solid phase extraction workflows.

[0067] Collected eluates 29 may be directed into sealed vials or microplate wells to reduce evaporative loss of volatile solvents 28 such as dichloromethane. In some cases, eluate 29 may be passed through inert filters to remove particulate matter or residual sorbent particles before further processing. Repeated elution fractions may be pooled to maximize recovery of y-tocopherol, and in some embodiments, antioxidant stabilizers may be added at trace levels to protect the analyte during this step. The use of controlled elution conditions ensures consistent recovery of y-tocophcrol and prepares the sample for the subsequent concentration and incubation steps.

[0068] At step 210, the method 200 includes evaporating the eluate 29 to dryness in order to form a residue 30. Evaporation may be carried out by applying a gentle stream of nitrogen gas 50 or argon 51 over the eluate 29 to displace volatile solvents 28 without excessive heating. Rotary evaporation under reduced pressure may also be used to efficiently remove large volumes of solvent 28, while centrifugal vacuum concentrators may be employed in moderate or small scale applications to achieve rapid drying under controlled conditions. ForAttorney Docket No. : 3229-6 PCT small volume samples, evaporation under a directed stream of nitrogen gas 50 is often preferred because it reduces the risk of oxidative degradation of y-tocopherol and preserves chromophoric integrity for subsequent reactions.

[0069] In certain embodiments, freeze drying may also be used when aqueous solvents 28 are present in the eluate 29 or when solvent 28 mixtures have both polar and non-polar components. In other embodiments, controlled temperature evaporation, such as use of heated dry block evaporators, may be applied to accelerate drying while maintaining sample integrity. The drying process may be monitored by visual inspection, gravimetric assessment, or automated sensors that detect solvent 28 vapor levels.

[0070] The residue 30 formed after evaporation typically contains concentrated y- tocopherol together with trace levels of co-extracted lipids, sterols, or minor impurities. In some embodiments, antioxidants such as butylated hydroxytoluene may be added in trace amounts during or immediately after drying to stabilize the residue 30. The drying step therefore provides a concentrated and solvent 28 free form of y-tocopherol that is ready for redissolution and subsequent derivatization with the reagent composition 31.

[0071] At step 212, the method 200 includes redissolving the residue 30 in the solvent 28 to prepare it for reaction with the reagent composition 31. Dichloromethane is often preferred as the solvent 28 because it provides ideal solubility for y-tocopherol, is compatible with the subsequent reagent composition 31, and offers high optical transparency within the visible wavelength range that includes the absorbance maxima of tocored and tocoyellow. Alternative solvents 28 may also be used, including chloroform, acetonitrile, ethyl acetate, tetrahydrofuran, or carefully selected mixtures of two or more solvents 28 to balance solubility, volatility, and stability of y-tocopherol.Attorney Docket No. : 3229-6 PCT

[0072] The volume of solvent 28 employed for redissolution may be tailored to the intended analytical configuration. For microplate spectrophotometers 16 or microfluidic systems, microliter quantities of solvent 28 may be sufficient, while larger milliliter volumes may be appropriate when conventional cuvettes or bench- scale spectrophotometers 16 are used. In certain embodiments, solvent 28 may be degassed or stored under inert atmosphere prior to use in order to reduce oxidative degradation of y-locophcrol or minimize background absorbance. In other embodiments, solvent 28 may contain trace stabilizing additives to further protect the analyte during handling and incubation.

[0073] The redissolution step provides a controlled and reproducible medium in which y- tocopherol is present in a solvent 28 environment optimized for subsequent derivatization, incubation, and spectrophotometric detection. By carefully selecting the solvent 28 and volume appropriate for the analytical platform, the method 200 maintains flexibility across laboratory, clinical, and industrial contexts while ensuring consistent performance.

[0074] At step 214, the method 200 includes mixing the redissolved residue 30 with a reagent composition 31 using the mixing device 10 to form a reaction mixture 32. The reagent composition 31 contains a nitrogen oxide producing chemical such as morpholinosydnonimine hydrochloride, which generates reactive nitrogen species that interact selectively with y-tocopherol. Alternative nitrogen oxide donors may also be used, including sodium nitroprusside, peroxynitrite generating systems, S-nitrosothiols such as S- nitrosoglutathione, nitrosyl complexes, or diazeniumdiolate salts. Selection of the nitrogen oxide donor may depend on the desired reaction rate, stability, and compatibility with the solvent 28.Attorney Docket No. : 3229-6 PCT

[0075] The nitrogen oxide donor is typically dissolved in methanol or another polar cosolvent such as ethanol 22, isopropanol, or acetonitrile. The final concentration of methanol or other polar co-solvent is generally maintained below about 10% of the total reaction mixture 32 in order to minimize solvent 28 interference with subsequent spectrophotometric detection while preserving solubility of the donor compound. In some embodiments, the nitrogen oxide donor may be prepared in advance as a stock solution and stored under cold or inert conditions to preserve stability prior to use.

[0076] Additional additives may also be included in the reagent composition 31 to refine reaction selectivity and minimize unwanted side reactions. These may include buffers to maintain pH stability, antioxidants at trace levels to prevent oxidative degradation of y- tocopherol during handling, or chelating agents to sequester transition metals that could otherwise catalyze secondary oxidation reactions. In certain embodiments, inert salts or ionic strength modifiers may be added to control solvent 28 interactions and enhance reproducibility.

[0077] The mixing device 10 ensures thorough dispersion of the reagent composition 31 into the redissolved residue 30, thereby forming a uniform reaction mixture 32 that is properly conditioned for incubation in step 216. By providing flexibility in donor selection, solvent 28 use, and stabilizing additives, this step allows the method 200 to be adapted to a wide range of analytical and operational settings.

[0078] At step 216, the method 200 includes incubating the reaction mixture 32 within incubator 15. The incubator 15 may include a water bath, a dry heating block, a thermostated chamber, a microplate heating device, or a temperature controlled reaction block. Incubation is typically performed at about 45 °C for a duration between about 10 and about 14 hours.Attorney Docket No. : 3229-6 PCTHowever, alternative temperature ranges between about 30°C and about 60°C and incubation periods ranging from about 2 hours to about 24 hours may also be used depending on sample throughput, stability, and desired reaction kinetics. In certain embodiments, accelerated heating methods may be applied, including microwave irradiation, infrared heating, or ultrasonic agitation, to shorten incubation time while maintaining the chemical conversion pathway.

[0079] The incubation is preferably carried out in the absence of light to minimize photodegradation or photochemical side reactions. Light exclusion may be achieved by using darkened incubation chambers, foil wrapping around individual vessels 18, or amber colored vessel 18 containers. The vessel 18 (FIG. 1) itself may be a sealed vial, a test tube, a microplate well, or a microfluidic reaction chamber, selected based on available equipment and sample volume. During this controlled incubation step, y-tocopherol undergoes chemical transformation through interaction with reactive nitrogen oxide species, leading to the formation of distinct chromophoric reaction products 17.

[0080] The chromophoric reaction products 17 include tocored, chemically identified as 2,7,8-trimethyl-2-(4,8,12-trimethyltridecyl)-5,6-chromaquinone, and tocoyellow, chemically identified as 2,7,8-trimethyl-2-(4,8,12-trimethyltridecyl)-5-nitro-6-chromanol. These compounds absorb strongly in the visible spectrum, particularly in the range of about 420 nanometers to about 450 nanometers, thereby producing a measurable absorbance signal that directly reflects the original y-tocopherol content. Additional nitrated, oxidized, or quinone related derivatives may also form under some reaction conditions depending on the nitrogen oxide donor and solvent 28 system employed. These alternative products may likewiseAttorney Docket No. : 3229-6 PCT provide chromophoric signatures that serve as indicators of y-tocopherol presence and concentration.

[0081] At step 218, the method 200 includes measuring absorbance of the reaction mixture 32 using the spectrophotometer 16. The spectrophotometer 16 may be configured as a single beam optical instrument, a dual beam optical instrument, a diode array detector, or other optical platforms designed for visible wavelength absorbance measurements. In laboratory environments, bench scale spectrophotometers with cuvette holders may be used, while microplate spectrophotometers allow analysis of multiple samples in parallel. In clinical or field settings, portable handheld devices, smartphone coupled photometers, or disposable strip readers may function as the spectrophotometer 16 to support point of care or decentralized testing.

[0082] Absorbance is typically measured within the visible wavelength range of about 420 nanometers to about 450 nanometers, with a preferred measurement at about 435 nanometers when dichloromethane is selected as the solvent 28. The specific wavelength selected may be adjusted depending on the solvent system and the relative contributions of tocored and tocoyellow to the absorbance spectrum.

[0083] The reaction mixture 32 may be analyzed using different container formats depending on instrument design and throughput. These formats may include quartz or plastic cuvettes, wells in a multiwell plate, sealed glass vials, or flow cells designed for continuous sample monitoring. In certain embodiments, optical path length adjustments may be made to optimize signal intensity, with shorter path lengths used for higher concentration reaction mixtures 32 and longer path lengths applied to more dilute preparations. The absorbance data may be captured digitally, stored in laboratory information management systems, orAttorney Docket No. : 3229-6 PCT transmitted wirelessly in field applications, ensuring integration with calibration data for subsequent quantitative analysis.

[0084] At step 220, the method 200 includes correlating the measured absorbance to the y-tocopherol concentration in the biological fluid 21. Correlation is performed by comparing the absorbance values obtained from the reaction mixture 32 to a calibration curve that is generated from authentic y-locophcrol standards processed through the same extraction, derivatization, incubation, and detection workflow. This ensures that matrix effects and procedural variations are accounted for in the calibration.

[0085] The calibration curve may be constructed across a broad concentration range of y- tocopherol, with standards prepared at multiple points to provide robust coverage for both low-level and high-level measurements. The calibration relationship may be linear in some ranges, while non-linear models may be used when higher concentrations introduce deviations due to instalment response or saturation effects. To improve reproducibility and minimize variability, internal standards such as 5-tocopherol, a-tocopherol, or structurally related analogs may be included in the workflow and analyzed in parallel.

[0086] Correlation may be refined through the use of advanced computational methods. These may include linear or polynomial regression, non-linear curve fitting, multivariate calibration approaches, or machine learning algorithms that can adapt to subtle variations in absorbance profiles. Statistical tools may be applied to assess the precision and accuracy of the calibration model, including calculation of coefficients of determination, residuals, and prediction intervals. In some embodiments, calibration data may be stored electronically and updated periodically with new standard runs to ensure long-term consistency across different instruments and laboratories.Attorney Docket No. : 3229-6 PCT

[0087] FIG. 3 illustrates system 300 for spectrophotometric detection of y-tocopherol in a biological fluid 21 or in a general sample 11. The system 300 includes the cartridge 27, which may contain silica gel 40 or other sorbent materials designed to isolate v-tocophcrol from the biological fluid 21. In some embodiments, the cartridge 27 may be omitted when direct analysis of a sample 11 is desired, such as when the sample 11 includes purified tocopherol standards, processed oils, fortified beverages, or extracts that do not require solid phase cleanup. In other embodiments, alternative sorbent formats may be used, including alumina, polymeric reversed phase resins, ion exchange media, or mixed mode cartridges that combine hydrophobic and ionic retention mechanisms.

[0088] The system 300 further includes the reagent composition 31, which contains morpholinosydnonimine hydrochloride dissolved in methanol and combined with the solvent 28, such as dichloromethane. Other nitrogen oxide producing chemicals may also be selected for the reagent composition 31, including nitrosothiols such as S -nitrosoglutathione, sodium nitroprusside, diazeniumdiolate salts, or peroxynitrite generating systems, thereby allowing flexibility in laboratory implementation and adaptation to reagent composition 31 availability. The reagent composition 31 may be pre-packaged in sealed ampules, vials, foil pouches, or lyophilized powder to improve stability during storage and shipping. The solvent 28 may include dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, toluene, or mixtures of these solvents, each providing an effective medium depending on the polarity and solubility characteristics of the sample 1 1 .

[0089] The system 300 further includes vessel 18 configured to hold the sample 11 or the eluate 29 containing y-tocopherol and to receive the reagent composition 31 for formation of the reaction mixture 32. The vessel 18 may take a variety of forms, including sealed glassAttorney Docket No. : 3229-6 PCT vials, cuvettes, disposable plastic tubes, or wells in a microplate. In some embodiments, the vessel 18 is provided as a single use component to reduce contamination, while in other cases reusable glass or quartz vessels are used in laboratory scale experiments.

[0090] The incubator 15 is included to maintain controlled temperature and light conditions during incubation of the reaction mixture 32. The incubator 15 may include a water bath, a dry block heater, a microplate incubator, or an enclosed temperature regulated cabinet, all of which maintain uniform heating across the vessel 18. In certain embodiments, advanced heating modalities such as microwave irradiation, infrared energy, or ultrasonic agitation may be integrated into the incubator 15 to accelerate formation of chromophoric reaction products 17.

[0091] The spectrophotometer 16 is provided to measure absorbance of the reaction mixture 32 at a wavelength typically within the visible range of about 420 to about 450 nanometers, and preferably at about 435 nanometers when dichloromethane is used as the solvent 28. The spectrophotometer 16 may include a bench scale instrument configured with a cuvette holder, a multiwell plate reader for parallel sample analysis, a portable field spectrophotometer, or a miniaturized device integrated with smartphone based optical sensors. In some embodiments, the spectrophotometer 16 may incorporate internal calibration software, data logging functions, wireless connectivity, or modules that automatically generate calibration curves from standard data. The spectrophotometer 16 is used to compare measured absorbance values against a calibration curve prepared from authentic y-tocopherol standards, allowing quantitative determination of y-tocopherol across both biological fluids 21 and other sample types 11.Attorney Docket No. : 3229-6 PCT

[0092] In certain embodiments, the system 300 may be supplied as a kit that includes premeasured solvents 28, disposable cartridges 27, the reagent composition 31 packaged in sealed vials or lyophilized format, one or more vessel 18 containers, and detailed instructions for use. The kit may also include authentic y-tocophcrol calibration standards in pre-dosed concentrations to simplify preparation of calibration curves. Optional accessories may include disposable pipettes, syringes, solvent reservoirs, or filters to streamline workflow in clinical or field settings. In some embodiments, the system 300 may be integrated into automated robotic platforms that perform sequential extraction, elution, reaction, and detection steps, thereby supporting high volume analysis in industrial or epidemiological applications.

[0093] In industrial contexts, the system 300 may be integrated into quality control workflows for edible oils, dietary supplements, or fortified food products to ensure antioxidant levels are maintained. In clinical contexts, the system 300 may be configured as a point of care diagnostic tool that requires minimal operator training, using simplified workflows that combine extraction, derivatization, and detection in a single cartridge 27 or microfluidic chip. In research contexts, the system 300 may be designed to support both method 100 and method 200, allowing investigators to either directly analyze simplified or purified samples 11 or to process more complex biological fluids 21 through extraction and cleanup with the cartridge 27 prior to reaction and detection.

[0094] The method 100, the method 200, and the system 300 together provide a structured and adaptable framework for detecting and quantifying y-tocopherol across diverse sample 1 1 types (e.g., biological fluids 21) and analytical environments. By supporting both direct solvent (13, 28) based workflows and extraction based workflows, the system 300 ensuresAttorney Docket No. : 3229-6 PCT that y-tocopherol can be measured with accuracy, reproducibility, and flexibility whether in laboratory research, clinical diagnostics, industrial production, or field monitoring programs.

[0095] FIG. 4 illustrates a controller 400 configured to manage and coordinate operation of the spectrophotometric detection system 300, including preparation steps of method 100 and method 200, as well as subsystems such as the mixing device 10, vessel 18, incubator 15, cartridge 27, and spectrophotometer 16. The controller 400 includes a processor 420 connected to a computer-readable storage medium or memory 430. The computer-readable storage medium or memory 430 can be a volatile type of memory, e.g., RAM, or a non-volatile type of memory, e.g., flash media, disk media, etc. In various aspects of the disclosure, the processor 420 can be another type of processor, such as a digital signal processor, a microprocessor, an ASIC, a graphics processing unit (GPU), a field-programmable gate array (FPGA) 450, or a central processing unit (CPU). In certain aspects of the disclosure, network inference or algorithmic processing can also be accomplished in systems that have weights implemented as memristors, chemically, or through other nontraditional inference calculations, as opposed to conventional processors. The memory 430 can be random access memory, read-only memory, magnetic disk memory, solid-state memory, optical disc memory, and / or another type of memory. In some aspects of the disclosure, the memory 430 can be separate from the controller 400 and can communicate with the processor 420 through communication buses of a circuit board and / or through communication cables such as serial ATA cables or other types of cables. The memory 430 includes computer-readable instructions that are executable by the processor 420 to operate the controller 400. In other aspects of the disclosure, the controller 400 can include a network interface 440 to communicate with other computers or to a server. A storage device 410 can be used for storingAttorney Docket No. : 3229-6 PCT calibration data, absorbance spectra, and concentration outputs. The disclosed method can run on the controller 400 or on a user device, including, for example, on a mobile device, an loT device, or a server system.

[0096] In some embodiments, the controller 400 is configured to monitor operating parameters of the system 300, including incubation temperature in the incubator 15, mixing duration and intensity of the mixing device 10, solvent (13, 28) composition in the vessel 18, and detection wavelength settings of the spectrophotometer 16. The controller 400 may control pumps, valves, or robotic pipettors for dispensing the sample 11, reagent composition (12, 31), and solvents (13, 28) into the vessel 18. It may further regulate separation and elution processes involving the cartridge 27 and schedule incubation intervals in the incubator 15. The controller 400 can also execute algorithms for calibration curve generation, background subtraction, noise reduction, peak integration, and y-locophcrol concentration correlation. In certain embodiments, the control system can be distributed, with separate modules networked across multiple devices (e.g., spectrophotometer 16, incubator 15, or mixing device 10), all connected by a high-speed data bus or wireless communication path with redundancy for failsafe operation.

[0097] In certain embodiments, the controller 400 can include or be operatively coupled to a non-transitory computer-readable medium storing computer-executable instructions which, when executed by one or more processors of the system 300, cause the processors to carry out coordinated control of sample 1 1 handling, derivatization, incubation, detection, and correlation steps. The instructions can cause the controller 400 to utilize actuators, mixing elements, heating modules, and optical detectors to selectively adjust: (i) reagent composition (12, 31) delivery into the vessel 18; (ii) incubation parameters in the incubator 15, includingAttorney Docket No. : 3229-6 PCT temperature, duration, and light exclusion; and (iii) optical readout conditions of the spectrophotometer 16, including wavelength range and integration time. By controlling these parameters in response to sensor inputs such as temperature probes, liquid level detectors, or optical reference standards, the controller 400 can dynamically adjust processing conditions to ensure accuracy and reproducibility. The controller 400 may also execute algorithms that receive real-time data from sensors embedded in the system 300 and apply this data to modulate operational variables such as mixing duration, solvent (13, 28) ratios, or spectrophotometer 16 calibration. Additional control functions can include scheduling absorbance measurements, directing data logging to the storage device 410, transmitting results via the network interface 440, and automatically applying calibration profiles to generate quantitative y-tocopherol concentrations across diverse sample types 11 and biological fluids 21.

[0098] Certain embodiments of the present disclosure may include some, all, or none of the above advantages and / or one or more other advantages readily apparent to those skilled in the art from the drawings, descriptions, and claims included herein. Moreover, while specific advantages have been enumerated above, the various embodiments of the present disclosure may include all, some, or none of the enumerated advantages and / or other advantages not specifically enumerated above.

[0099] The embodiments disclosed herein are examples of the disclosure and may be embodied in various forms. For instance, although certain embodiments herein are described as separate embodiments, each of the embodiments herein may be combined with one or more of the other embodiments herein. Specific structural and functional details disclosed herein are not to be interpreted as limiting, but as a basis for the claims and as a representative basisAttorney Docket No. : 3229-6 PCT for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure. Like reference numerals may refer to similar or identical elements throughout the description of the figures.

[0100] The phrases “in an embodiment,” “in embodiments,” “in various embodiments,” “in some embodiments,” or “in other embodiments” may each refer to one or more of the same or different example embodiments provided in the present disclosure. A phrase in the form “A or B” means “(A), (B), or (A and B).” A phrase in the form “at least one of A, B, or C” means “(A); (B); (C); (A and B); (A and C); (B and C); or (A, B, and C).”

[0101] It should be understood that the foregoing description is only illustrative of the present disclosure. Various alternatives and modifications can be devised by those skilled in the art without departing from the disclosure. Accordingly, the present disclosure is intended to embrace all such alternatives, modifications, and variances. The embodiments described with reference to the attached drawing figures are presented only to demonstrate certain examples of the disclosure. Other elements, steps, methods, and techniques that are insubstantially different from those described above and / or in the appended claims are also intended to be within the scope of the disclosure.

Claims

Attorney Docket No. : 3229-6 PCTWHAT IS CLAIMED IS:

1. A method for spectrophotometric detection of y-tocopherol in a sample, comprising: mixing the sample with a reagent composition in a solvent to form a mixture; incubating the mixture; measuring absorbance of the mixture; and correlating the measured absorbance to v-tocophcrol concentration in the sample.

2. The method of claim 1, further comprising incubating the mixture at about 45°C for about 8 to about 10 hours.

3. The method of claim 1, further comprising measuring the absorbance of the mixture at a wavelength in the range of about 420 nm to about 450 nm.

4. The method of claim 1, wherein the reagent composition comprises a nitrogen oxide-producing chemical.

5. The method of claim 4, wherein the nitrogen oxide-producing chemical comprises morpholino sydnonimine hydrochloride .

6. The method of claim 5, further comprising dissolving the morpholinosydnonimine hydrochloride in methanol such that the methanol constitutes no more than about 10% by volume of the mixture.Attorney Docket No. : 3229-6 PCT7. The method of claim 1 , wherein the solvent comprises dichloromethane.

8. The method of claim 1, further comprising incubating the mixture at about 45 °C for about 8 to about 1 hours in the absence of light.

9. The method of claim 1, further comprising forming a chromophoric reaction product comprising at least one of:2.7.8-trimethyl-2-(4, 8 , 12-trimethyltridecyl)-5 ,6-chromaquinone (tocored) ; or2.7.8-trimethyl-2-(4,8,12-trimethyltridecyl)-5-nitro-6-chromanol (tocoyellow).

10. The method of claim 4, further comprising generating a calibration curve by treating a series of concentrations of authentic ' / -tocopherol with the nitrogen oxide-producing chemical.

11. The method of claim 1, further comprising, prior to the mixing step, combining the sample with ethanol to form a pre-extraction mixture.

12. The method of claim 11, further comprising extracting the pre-extraction mixture with hexane to form a hexane layer.

13. The method of claim 12, further comprising subjecting the hexane layer to solid-phase extraction on a cartridge.

14. The method of claim 13, further comprising eluting / -tocopherol from the cartridge with aAttorney Docket No. : 3229-6 PCT mixture of hexane and a solvent to obtain an eluate.

15. The method of claim 14, further comprising evaporating the eluate to dryness and redissolving a residue resulting from the evaporating in the solvent.

16. The method of claim 1, wherein the sample comprises a biological fluid, a food product, a dietary supplement, or a plant extract.

17. The method of claim 13, wherein the cartridge is a silica gel cartridge.

18. The method of claim 1, further comprising incubating the mixture at about 45°C for about 10 to about 14 hours in the absence of light.

19. A method for spectrophotometric detection of / -tocopherol in a biological fluid, comprising: mixing the biological fluid with ethanol in a vessel using a mixing device to form a mixture; extracting the mixture with hexane to form a hexane layer; subjecting the hexane layer to solid-phase extraction on a cartridge; eluting / -tocopherol from the cartridge with a mixture of hexane and a solvent to derive an eluate; evaporating the eluate to dryness; redissolving formed residue in the solvent; mixing the redissolved formed residue with a reagent composition using a mixing device to form a reaction mixture;Attorney Docket No. : 3229-6 PCT incubating the reaction mixture; measuring absorbance of the reaction mixture using a spectrophotometer; and correlating the measured absorbance to y-tocopherol concentration in the biological fluid using a calibration curve generated from authentic y-tocopherol standards.

20. A system for spectrophotometric detection of y-tocopherol in a sample, comprising: a solid-phase extraction cartridge comprising silica gel configured to isolate y-tocopherol from the sample; a vessel configured for incubation of a mixture at about 45 °C for about 10 to about 14 hours in the absence of light to produce tocored and tocoyellow, the mixture formed by a reagent composition comprising morpholino sydnonimine hydrochloride dissolved in methanol and combined with dichloromethane; and a spectrophotometric device configured to measure absorbance of the mixture at about 435 nm and determine y-tocopherol concentration using a calibration curve prepared from authentic y- tocopherol standards, wherein the solid-phase extraction cartridge is configured to isolate y-tocopherol from the sample and deliver the isolated y-tocopherol for incubation in the vessel.

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