TW201905451A - Analysis method for vitamins and their stereoisomers achieving the purpose of improving the sample detection efficiency - Google Patents

Abstract

This invention provides an analysis method for vitamins and stereoisomers thereof, which utilizes a sample pretreatment step of extracting vitamins and their stereoisomers in a sample by using a high-performance liquid chromatography tandem fluorescence detector and an ultraviolet-visible light detector detection system with metaphosphoric acid as an extract. The pretreatment step of the sample to be tested is effectively simplified, and vitamin B2 and vitamin C and their stereoisomers are separated with trifluoroacetic acid and cyanomethane and their stereoisomers as mobile phase chromatography, which significantly improves the efficiency of analysis and detection of the vitamins and their stereoisomers, so that vitamins and their stereoisomers in commercially available nutritional supplements may be quickly separated and detected, achieving the purpose of improving the sample detection efficiency.

Description

Analytical method for vitamins and their stereoisomers

The invention relates to a method for analyzing vitamins, in particular to a method for detecting and analyzing vitamins in a sample to be tested by liquid chromatography and optical detection techniques.

High performance liquid chromatography (HPLC) mainly uses the interaction between the stationary phase in the chromatography column and the mobile phase flowing in the chromatography column to change the difference in the mixture entering the chromatography column. The rate at which the constituent materials flow in the chromatography column, thereby separating the different constituent materials in the mixture, and causing the different constituent materials in the mixture to flow out from the chromatography column at different specific time points, thereby The different components of the substance are separated.

The Fluorescence Detector and the UV-VIS Detector are common optical inspection instruments. The fluorescence detector can mainly perform qualitative and quantitative detection on a substance capable of emitting fluorescence. The fluorescence detector first provides excitation light of a specific wavelength to excite a specific substance to generate fluorescence emission, and then measures a specific wavelength. The underlying fluorescent exposure gives qualitative and quantitative results for that particular substance. The ultraviolet-visible light detector uses light of a specific wavelength in the ultraviolet to visible wavelength range to strike a specific substance, and the specific substance is obtained by the difference in energy generated by the specific wavelength of light passing through the specific substance. The absorption spectrum is then used to qualitatively and quantitatively analyze the specific substance.

In the past, the analysis of different substances in the mixture generally required the sample to pass through complex pre-treatment steps. For example, before analyzing the vitamin B2 in the sample, the sample of vitamin B2 should be weighed before adding the concentration to the sample. After 0.1 M sulfuric acid, it was heated in a water bath at 90 ° C, and shaken every 5 minutes to make the acid solution and the sample fully act. After heating for 30 minutes, take out the sample to be tested and wait for it to cool, then add 2 ml of sodium acetate at a concentration of 4 M, and adjust the pH to a range of 4.5 to 4.7 to precipitate the protein, quantify it to the mark, and filter it. After that, the pretreatment steps of the sample to be tested for vitamin B2 can be completed. The pretreatment of the entire vitamin B2 takes about 4 to 5 hours from waiting for the heater to heat up to 90 ° C until the sample is cooled to adjust the pH.

In addition, vitamin C and its stereoisomers are stereoisomers of each other. If the solution is chromatographed in a KH ₂ PO ₄ solution having a pH of 2.5 and pumped at a constant rate, the analysis time takes about 15 minutes. If the vitamin B2 is subjected to a gradient elution with sodium hexanesulfonate (PICB6) and cyanomethane, the analysis time is about 30 minutes. Therefore, if the chromatographic separation of vitamin C and its stereoisomers and vitamin B2 cannot be performed simultaneously, it takes at least 45 minutes to complete the chromatographic separation of vitamin C and its stereoisomers and vitamin B2. Such a detection process is cumbersome and time consuming, and the operator needs to perform a sub-operation to detect the vitamin B2 and vitamin C in the sample, so the slow detection efficiency can not meet the current market demand.

In addition, in recent years, people's emphasis on food safety has gradually increased. Whether it is the correctness of the labeling of food contents or the toxicity of harmful substances, the demand for food detection has been greatly increased. Therefore, how to improve the efficiency of food testing is also the goal that is constantly being sought by all walks of life, in the hope of developing a faster and more efficient detection and analysis method.

In view of the problem of poor detection efficiency of the prior art, the object of the present invention is to improve the efficiency of analysis and detection. By simplifying the complicated sample pre-processing steps, the new detection system can simultaneously analyze and detect two or more target substances. And the sample can automatically and continuously detect different target substances in the same sample between different detecting instruments, thereby saving the time and labor and improving the detection efficiency.

In order to achieve the foregoing object, the present invention provides a method for analyzing a vitamin and a stereoisomer thereof, wherein the method comprises: providing a sample to be tested, the sample to be tested containing vitamin B2, vitamin C and erythorbic acid; The sample is conveyed into a detection system of a high-performance liquid chromatograph tandem fluorescence detector and an ultraviolet-visible light detector to perform chromatographic separation and optical detection on the sample to be tested.

The invention utilizes a high-performance liquid chromatography tandem fluorescence detector and an ultraviolet-visible light detector to construct a set of analysis and detection system for vitamins and stereoisomers thereof, and the analysis detection system can be connected in series by analyzing pipelines The high-performance liquid chromatograph is coupled with a fluorescent detector and an ultraviolet-visible light detector. The analysis and detection system only needs to input the sample to be tested into a high-performance liquid chromatograph for separation. Automatically transport the separated sample to be tested into a fluorescent detector or an ultraviolet-visible detector for optical detection, thereby eliminating the transfer of the sample to be tested between the high performance liquid chromatograph and the optical detector. It also requires the intervention of human intervention, reducing the risk of sample contamination caused by manual operation, and at the same time saving the time required for detection and analysis by continuous continuous work between chromatographic separation and optical detection.

Preferably, the analysis method comprises the step of determining a standard product before the sample to be tested is measured. The assay step of the standard comprises: providing a sample of a standard containing a known concentration of vitamin B2, a known concentration of vitamin C, and a known concentration of erythorbic acid; the standard sample is delivered to the high performance fluid The phase chromatograph is connected to the fluorescence detector and the ultraviolet-visible light detector detection system to perform chromatographic separation and optical detection on the standard sample; the peak area of the known concentration of vitamin B2 obtained by optical detection The peak area of the known concentration of vitamin C and the peak area of the known concentration of erythorbic acid were used to calculate the contents of vitamin B2, vitamin C and erythorbic acid in the sample to be tested. The technical means first uses the known concentration of the standard sample to obtain the peak concentration corresponding to the known concentration of vitamin B2, the known concentration of vitamin C, and the known concentration of erythorbic acid. The peak area of the spectrum measured by the unknown concentration of vitamin B2, vitamin C and erythorbic acid in the sample is used to derive the true concentration of vitamin B2, vitamin C and erythorbic acid in the sample to be tested.

Preferably, the analysis method comprises: extracting an untreated sample to be tested by using 3% to 10% by weight of metaphosphoric acid to obtain a mixed liquid, and the untreated sample contains vitamin B2. Vitamin C and erythorbic acid; the mixed liquid is centrifuged, and the supernatant is collected to obtain the sample to be tested. The technical means utilizes a simplified pre-processing step of the sample to be tested, and the pre-treated sample to be tested can be transported into the analytical detection system of the present invention for chromatographic separation by extracting and centrifuging the sample to be tested by the metaphosphoric acid solution. And optical inspection, eliminating the need for complex sample pre-processing steps in the past, including: sample and acid solution configuration, water bath heating, natural cooling, and pH adjustment, reducing the time required for sample preparation steps. Reduce the time required for probing analysis.

More preferably, the centrifugation speed condition is from 2500 rpm to 3500 rpm.

Preferably, the high performance liquid chromatograph is provided with a reverse phase column, and the analysis method comprises: dissolving the sample to be tested in a mobile phase solvent and injecting into the reverse phase column for chromatographic separation; Wherein the mobile phase solvent is a mixed solvent of trifluoroacetic acid and cyanomethane, and the mixing ratio of the mobile phase solvent trifluoroacetic acid and cyanomethane is 100:0 gradually adjusted to 80:20 with time, and the flow rate of the mobile phase solvent is set. It is 1.4 ml/min. The technical means can simultaneously dissolve the target substance to be detected in the present invention by using the mobile phase solvent: the characteristics of vitamin B2, vitamin C and erythorbic acid, so that the mobile phase solvent can be directly dissolved after simultaneously dissolving vitamin B2, vitamin C and erythorbic acid. It is injected into the reverse phase column for chromatographic separation, and by controlling the flow rate of the mobile phase solvent to assist in achieving the separation of vitamin B2, vitamin C and erythorbic acid in a short time.

Preferably, the analysis method comprises optically detecting vitamin B2 using a fluorescent detector, and optically detecting vitamin C and erythorbic acid using an ultraviolet-visible light detector. This technical means utilizes the optical properties of vitamin B2 in aqueous solution to emit greenish yellow fluorescent light, and the optical property is used to quantitatively detect the content of vitamin B2 by a fluorescent detector. The vitamin C and its stereoisomer, erythorbic acid, can absorb the energy of vitamin C and its stereoisomers through an ultraviolet-visible detector because it absorbs part of the energy that penetrates its own ultraviolet or visible light. The characteristics were tested, and the optical properties of the vitamin C and its stereoisomers were quantitatively detected by an ultraviolet-visible light detector.

In the following, the embodiments of the present invention will be described by the following specific examples, and those skilled in the art can readily understand the advantages and effects of the present invention, and without departing from the spirit of the present invention. Various modifications and changes are made to implement or apply the present invention.

The invention firstly utilizes a high-performance liquid chromatography tandem fluorescence detector and an ultraviolet-visible light detector to construct a set of vitamin and its stereoisomer analysis and detection system, so that different vitamins and their stereoscopic differences in the sample After being separated by a high-performance liquid chromatograph at different times, the structure can be directly detected into a fluorescent detector or an ultraviolet-visible detector for optical detection and calculated by optical detection of the optical spectrum. The content of the target to be tested contained in the sample, thereby simultaneously achieving qualitative and quantitative analysis of the vitamin and its stereoisomer in the sample, and the analysis and detection system of the vitamin and its stereoisomer can simultaneously The target to be tested is tested and analyzed.

The target to be tested of the present invention is mainly vitamin B2 and vitamin C and stereoisomers thereof. First, the high-performance liquid chromatography tandem fluorescence detector and the ultraviolet-visible light detector of the present invention are used to detect vitamin B2 and vitamin C and its stereoisomers (isoascorbic acid) standards, wherein the vitamin B2 The initial concentration is 10 ppm, and the standard solution is 0.1 to 1.35 ppm, and the initial concentration of the vitamin C and its stereoisomers is 500 ppm, which is then 25 to 125 ppm. The standard solution, followed by the standard solution of different concentrations of vitamin B2 and the standard solution of vitamin C and its stereoisomers are detected by a fluorescence detector and an ultraviolet-visible light detector, respectively, and the area calculated by integrating the waveform is recorded. As shown in Table 1 below, a linear regression curve of the standard of vitamin B2 and the standard of vitamin C and its stereoisomers was obtained after the completion of the test.

The mixed standard solution containing 10 μl of vitamin B2 and vitamin C and its stereoisomers is sent to a high performance liquid chromatography tandem fluorescence detector for chromatographic separation. The high performance liquid chromatography The chromatography column used in the instrument is a reverse phase column, and its static phase is commercially available Atlantisâ T3 5um 4.6*250mm. For liquid chromatography, the mobile solvent used is a mixed solvent of trifluoroacetic acid and cyanomethane in a volume percentage of 0.1%, and the mixing ratio is gradually increased from trifluoroacetic acid: cyanomethane to 100:0. The temperature was adjusted to 80:20, and the flow rate of the high performance liquid chromatograph was set to 1.4 ml/min, and the chromatographic separation was carried out by a gradient bank method. Herein, the use of the aforementioned mobile phase can facilitate simultaneous chromatography of vitamin C and isoascorbic acid.

The analysis results are shown in Fig. 1. When the above chromatographic conditions are used, the ultraviolet-visible light detector can obtain ultraviolet-visible absorption peaks of vitamin C and its stereoisomers at 4.112 and 4.431 minutes. The light-visible detector sets a wavelength of 245 nm. In order to obtain a linear regression curve of vitamin C and dystrophic blood concentration and waveform, a standard solution with a concentration between 25 and 125 ppm was prepared, and then detected by an ultraviolet-visible light detector, and the results are listed in Table 1 below. The area calculated by the integral represents the standard content of vitamin C and its stereoisomers in the standard. Table 1: Standard concentration of vitamin C and erythorbic acid, waveform area after detection, and linear regression curve.

Next, at 12.782 minutes, the fluorescence detector can obtain a fluorescent emission peak of vitamin B2. As shown in FIG. 2, the fluorescence detector sets an excitation wavelength of 447 nm. Also to obtain a linear regression curve of vitamin B2 concentration and waveform, a standard solution with a concentration between 0.1 and 1.35 ppm was prepared, and the emission wavelength detected by the fluorescence detector was 530 nm, and then detected by a fluorescent detector. Listed in Table 2 below, the area calculated by the integral represents the standard content of vitamin B2 in the standard. Table 2: Standard concentration of vitamin B2, waveform area after detection, and linear regression curve.

The high-performance liquid chromatography tandem fluorescence detector and the ultraviolet-visible light detector can simultaneously separate and optically detect the vitamin C and its stereoisomer standards and vitamin B2 standards. And establish a standard content of vitamin C and its stereoisomers and vitamin B2 by a known concentration of vitamin C and its stereoisomer standard and vitamin B2 standard, and then the standard content is unknown. Estimation criteria for the content of vitamin C and its stereoisomers and vitamin B2 in the sample to be tested.

After the above-mentioned established vitamin C and its stereoisomers and vitamin B2 content estimation standards, the samples to be tested of unknown concentration will be further tested below.

This example uses three commercially available nutritional supplements as the test sample (samples 1-1 to 1-7, samples 2-1 to 2-8, and samples 3-1 to 3-5, respectively) for vitamin C and vitamins. B2 content detection and analysis.

First, weigh 5 g of the sample to be tested and place it in a 25 ml brown volume bottle, and then add 5 ml of 10% by weight of metaphosphoric acid as an extract to the brown volume bottle. The vitamin C and vitamin B2 in the sample to be tested are extracted, and then added to a volume of 25 ml in deionized water, and then placed in a shaker to be homogeneously dissolved, and then the dissolved 25 ml solution is poured. The volume is 50 ml in a centrifuge tube for further centrifugation, wherein the centrifuge is set to 3000 rpm and the centrifugation time is set to 10 minutes. After the centrifugation is completed, the supernatant in the centrifuge tube is taken out and The aqueous filter membrane with a pore size of 0.45 μm is filtered, and the pre-treated sample to be tested can be obtained after filtration.

In the traditional food, the test method of vitamin C is to grind and mix the sample, take about 1 gram of solid sample, about 10 grams of liquid sample, accurately weighed, then add 2% of metaphosphoric acid solution 40 mL, vortex mixing 1 Minute, then make up to 50 mL with 2% solution of metaphosphoric acid, centrifuge at 4000×g for 10 minutes at 4°C, take 25 mL of supernatant, add 10 mL of 4% L-cysteine solution, mix. After homogenization, adjust to neutral with 20% trisodium phosphate solution, stir for 5 minutes, adjust the pH to 2.5 with 20% metaphosphoric acid solution, and then dilute to 50 mL with 2% solution of metaphosphoric acid. For use as a test fluid. However, the present invention only needs to use 10% of 5% phosphoric acid to centrifuge to obtain a clear liquid, which can save the dosage of the medicament and effectively shorten the pretreatment time.

Then, 10 μl of the pre-treated sample to be tested is sent to a high-performance liquid chromatography tandem fluorescence detector for chromatographic separation. The chromatography column used in the high-performance liquid chromatography is reversed. The phase column, the mobile phase used in the high performance liquid chromatography is a mixed solvent of trifluoroacetic acid and cyanomethane in a volume percentage of 0.1%, and the flow rate of the high performance liquid chromatography is set to 1.4. Ml/min, and chromatographic separation using a gradient bank. The reverse phase column is an Atlantis T3 column, which is a reversed-phase C18 column of cerium oxide. The Atlantis T3 column provides alternative selectivity and increased compound retention compared to conventional C18 columns. Because of the bonding density of C18 alkyl bond and end group capping technology, it has good peak shape and proper retention efficiency for all compounds, and has excellent chemical stability over a wide pH range, especially in Very stable in an acidic environment.

The analysis results are shown in Figure 3. The chromatographic column, mobile phase, flow rate setting, and levee method used in the high-performance liquid chromatograph are the same as those for the standard test of vitamin C, in Figure 3. The peak time point obtained by the ultraviolet-visible fluorescent detector at 4.139 minutes is similar to the peak time point of the aforementioned standard for detecting the vitamin C, which is 4.12 minutes, and the error value is less than 0.03 minutes; therefore, the ultraviolet light is The peak obtained by the visible light detector at 4.139 minutes represents the peak of vitamin C in the sample to be tested.

In order to detect the content of vitamin C in the sample to be tested, the ultraviolet light-visible light detector is also set to a wavelength of 245 nm, and the waveform area of the aforementioned peak is calculated by integrating, and the waveform area is substituted into the aforementioned vitamin C standard. The obtained linear regression curve can calculate the concentration of vitamin C in the sample to be tested, and further, the concentration can be converted into the number of grams of vitamin C contained per gram of the sample. The detailed results are shown in Table 3. The fluorescent detector then obtains a fluorescent emission peak at 12.768 minutes, as shown in FIG. Since the peak time point obtained by the fluorescence detector at 12.768 minutes is similar to the peak time point of the aforementioned standard for detecting the vitamin B2, which is 12.782 minutes, the error value is less than 0.03 minutes, and the high performance liquid chromatography is used. The chromatographic column, mobile phase, flow rate setting, and levee method used in the same are the same as those for the standard test of vitamin B2. Therefore, it can be estimated that the peak obtained by the fluorescent detector at 12.768 minutes represents The peak of vitamin B2 in the sample is measured.

In addition, in order to detect the content of vitamin B2 in the sample to be tested, the excitation wavelength set by the fluorescence detector is also 447 nm, and the emission wavelength detected by the fluorescence detector is also 530 nm, and the waveform area of the peak is calculated by integrating. By substituting the waveform area into the linear regression curve obtained by the regression of the vitamin B2 standard, the concentration of vitamin B2 in the sample to be tested can be calculated, and in addition, the concentration can be converted into the vitamin B2 per gram of the sample. The detailed results are listed in Table 3.

Further, by comparing the area calculated by the waveform with the estimated amount of the vitamin B2, the true content of the vitamin B2 in the sample to be tested can be calculated. Table 3: The weight of the sample to be tested for three commercially available nutritional supplements, the area of the waveform measured by an ultraviolet-visible light detector or a fluorescence detector, the calculated concentration and the amount per gram of sample.

Finally, the calculated actual content of vitamin C in the nutritional supplement of the sample to be tested and the true content of vitamin B2 are very similar to the labeled content of the commercially available nutritional supplement supply unit.

When participating in the test ability comparison, when the standard score (Z-score) is between -2 and 2, it can pass the test and meet the manufacturer's requirements. The analytical method of the vitamin C and vitamin B2 of the present invention is calculated by the Z-score. Between -1 and 1, the high-performance liquid chromatography tandem fluorescence detector and the ultraviolet-visible light detector detection and detection system used in the present invention and the pre-processing steps of the sample to be tested are indeed effective and Accurately detect the true content of vitamin C in the sample to be tested and the true content of vitamin B2.

In summary, the high-performance liquid chromatography tandem fluorescence detector of the present invention and the ultraviolet-visible light detector of the vitamin and its stereoisomer analysis and detection system and the treatment to enter the analysis and detection system The pretreatment method of the sample can not only accurately detect the true content of vitamin C and its stereoisomers in the sample to be tested, but also the true content of vitamin B2, and can be continuously in a very short time (within 15.5 minutes). Complete the actual content of vitamin C and its stereoisomers in the sample to be tested and the separation and quantitative detection of vitamin B2, thereby having the content component capable of simultaneously analyzing two or more nutritional supplements without complicated sample preparation steps. Detecting advantages, effectively improving the detection and analysis efficiency of the content components of nutritional supplements, and is suitable for use in laboratories for general routine analysis.

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BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a UV-visible light spectrum of a standard of vitamin C and its stereoisomers of the analysis method of the present invention. Fig. 2 is a fluorescence detection spectrum of a standard of vitamin B2 of the analysis method of the present invention. Fig. 3 is a spectrum of ultraviolet-visible light detection of a sample of vitamin C of the analysis method of the present invention. Fig. 4 is a fluorescence detection spectrum of a sample of vitamin B2 to be tested according to the analysis method of the present invention.

Claims (10)

A method for analyzing a vitamin and a stereoisomer thereof, the method comprising: providing a sample to be tested, the sample to be tested containing vitamin B2, vitamin C and erythorbic acid; and delivering the sample to be tested into high performance liquid chromatography The instrument is connected to the fluorescence detector and the ultraviolet-visible light detector to perform chromatographic separation and optical detection on the sample to be tested. The method for analyzing a vitamin and a stereoisomer thereof according to claim 1, wherein the method comprises: performing a measuring step of a standard before the measuring of the sample to be tested; and the measuring step of the standard comprises: providing a standard a sample of the standard containing a known concentration of vitamin B2, a known concentration of vitamin C, and a known concentration of erythorbic acid; the standard sample is delivered to a high performance liquid chromatography tandem fluorescence detector and UV In the detection system of the light-visible light detector, chromatographic separation and optical detection are performed on the sample of the standard; the peak area of the known concentration of vitamin B2 obtained by optical detection, the peak area of the known concentration of vitamin C, and The peak area of the known concentration of erythorbic acid is calculated as the content of vitamin B2, vitamin C and erythorbic acid in the sample to be tested. The method for analyzing a vitamin and a stereoisomer thereof according to claim 1 or 2, wherein the analysis method comprises: extracting an untreated sample to be tested by using 3% to 10% by weight of metaphosphoric acid, A mixed liquid is obtained, and the untreated sample to be tested contains vitamin B2, vitamin C and erythorbic acid; the mixed liquid is centrifuged, and the supernatant is collected to obtain the sample to be tested. The method for analyzing a vitamin and a stereoisomer thereof according to claim 3, wherein the centrifugal rotation speed condition is 2500 rpm to 3500 rpm. The method for analyzing a vitamin and a stereoisomer thereof according to claim 1 or 2, wherein the high performance liquid chromatograph is provided with a reverse phase column, and the analysis method comprises: dissolving the sample to be tested The mobile phase solvent is injected into the reverse phase column for chromatographic separation; wherein the mobile phase solvent is a mixed solvent of trifluoroacetic acid and cyanomethane, and the mixing ratio of the mobile phase solvent trifluoroacetic acid and cyanomethane is between 100: The flow rate of the mobile phase solvent was set from 1.35 ml/min to 1.45 ml/min between 0 and 80:20. The method for analyzing a vitamin and a stereoisomer thereof according to claim 3, wherein the high performance liquid chromatograph is provided with an inverse phase column, and the analysis method comprises: dissolving the sample to be tested in a mobile phase The solvent is injected into the reverse phase column for chromatographic separation; wherein the mobile phase solvent is a mixed solvent of trifluoroacetic acid and cyanomethane, and the mixing ratio of the mobile phase solvent trifluoroacetic acid and cyanomethane is between 100:0. The flow rate of the mobile phase solvent was set between 1.35 ml/min and 1.45 ml/min between 80:20. The method for analyzing a vitamin and a stereoisomer thereof according to claim 4, wherein the high performance liquid chromatograph is provided with a reverse phase column, and the analysis method comprises: dissolving the sample to be tested in a mobile phase The solvent is injected into the reverse phase column for chromatographic separation; wherein the mobile phase solvent is a mixed solvent of trifluoroacetic acid and cyanomethane, and the mixing ratio of the mobile phase solvent trifluoroacetic acid and cyanomethane is between 100:0. The flow rate of the mobile phase solvent was set between 1.35 ml/min and 1.45 ml/min between 80:20. The method for analyzing a vitamin and a stereoisomer thereof according to claim 1 or 2, wherein the analysis method comprises optically detecting vitamin B2 using a fluorescent detector, and using an ultraviolet-visible light detector for vitamin C and different Ascorbic acid was optically detected. The method for analyzing a vitamin and a stereoisomer thereof according to claim 3, wherein the analysis method comprises optically detecting vitamin B2 using a fluorescent detector, and performing vitamin C and erythorbic acid using an ultraviolet-visible light detector. Optical inspection. The method for analyzing a vitamin and a stereoisomer thereof according to claim 5, wherein the analysis method comprises optically detecting vitamin B2 using a fluorescent detector, and performing vitamin C and erythorbic acid using an ultraviolet-visible light detector. Optical inspection.