Determination Of Niacin Research Articles

Immunological strip sensor for the rapid determination of niacin in dietary supplements and foods.

Herein, four haptens of niacin (Vitamin B3, VB3) were designed, and after a series of experiments, it was concluded that hapten D had the best immune effect. To avoid false positives in the detection of real samples, a monoclonal antibody (mAb) against VB3 was prepared by a matrix effect-enhanced mAb screening method. The concentration of the inhibition rate reaching 50% (IC50) was 603.41 ng mL-1 and the limit of detection (LOD) using an indirect enzyme-linked immunosorbent assay (ic-ELISA) was 54.89 ng mL-1. A lateral flow immunochromatographic assay (LFIA) based on gold nanoparticles was established to detect the concentration of VB3 in compound vitamin B tablets and infant formulas, with a visual LOD of 5 μg mL-1. Using a handheld reader, the quantitative LOD was calculated to be 0.60 μg mL-1. The contents of the compound vitamin B tablets and infant formulas were also verified by liquid chromatography. Therefore, the LFIA developed in this study can be applied to the specific identification and rapid detection of niacin in nutritional dietary supplements, thus meeting the market's demand for efficient niacin detection methods.

Simultaneous determination of niacin and pyridoxine at trace levels by using diode array high-performance liquid chromatography and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry.

A highly sensitive and simple diode-array high-performance liquid chromatography and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry method was developed for the simultaneous determination of niacin and pyridoxine in pharmaceutical drugs, tap water, and wastewater samples. To determine the in vivo behavior of niacin and pyridoxine, analytes were subjected to simulated gastric conditions. The calibration plots of the diode-array high-performance liquid chromatography and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry method showed good linearity over a wide concentration range with close to 1.0 correlation coefficients for both analytes. The limit of detection/limit of quantitation values for liquid chromatography quadrupole time-of-flight tandem mass spectrometry analysis were 1.98/6.59 and 1.3/4.4 μg/L for niacin and pyridoxine, respectively, while limit of detection/limit of quantitation values for niacin and pyridoxine in high-performance liquid chromatography analysis were 3.7/12.3 and 5.7/18.9 μg/L, respectively. Recovery studies were also performed to show the applicability of the developed methods, and percentage recovery values were found to be 90-105% in tap water and 94-97% in wastewater for both analytes. The method was also successfully applied for the qualitative and quantitative determination of niacin and pyridoxine in drug samples.

Development of a chromatographic low pressure flow injection system using amperometric detection: Application to the analysis of niacin in coffee

In this work, an analytical flow system able to perform low pressure chromatography with amperometric detection is presented. As case study, the determination of niacin (vitamin B3) in coffee brewed samples was selected. The manifold comprised a 1.0cm length monolithic column coated with didecyldimethylammonium bromide, a laboratory-made boron doped diamond electrode, and featured in-line ionic strength adjustment of the mobile phase. The figures of merit concerning the selected case study namely, detection limit, 7.90×10−7M, determination rate, ca. 10 samplesh−1, mobile phase and ISA solution consumption, ca. 2.6mL per analysis, and CV, below 5% for retention time and peak height, showed the competitiveness of this analytical strategy comparing to the described HPLC methods for niacin determination. The strategy displays a simple configuration, low cost, fast and easy assembling, foreseeing its use to general purpose applications.

Determination of niacin and its metabolites using supercritical fluid chromatography coupled to tandem mass spectrometry.

Niacin, a water-soluble vitamin belonging to the vitamin B group, has been known to cause various problems in the human body when deficient. The vitamin is derived from the diet and afterwards, niacin and its metabolites are secreted in blood or urine. It can be analyzed using liquid chromatography (LC) coupled to mass spectrometry, but niacin and its metabolites are very polar compounds. Recently, supercritical fluid chromatography (SFC) is gaining attention for polar compound analysis. To our best knowledge, the report on the analysis of endogenous-very hydrophilic metabolites in biofluids by SFC has not been found. In this study, we investigated whether the separation of hydrophilic metabolites in biofluids is achievable by SFC. In addition, we also examined the applicability of SFC coupled to MS in extrapolating unknown metabolites by means of spectra information. As a result, an analysis method to quantify the target compounds using SFC/MS/MS was constructed for niacin and its metabolites. Additional putative metabolites from niacin were also identified using the MS fragmentation spectra in plasma and urine. Consequently, the method using SFC/MS/MS allowed for the analysis of polar compounds with low log P ranging from -3.7 to 0.29. This study is the first report of the separation of niacin and its seven metabolites in human urine and these results showed that SFC-MS/MS can be an alternative technique for hydrophilic metabolite analysis.

Investigation of isotope dilution mass spectrometric (ID-MS) method to determine niacin in infant formula, breakfast cereals and multivitamins

An isotope dilution LC/mass spectrometric (ID-LC/MS) method was developed as a candidate reference method for the accurate determination of niacin in infant formula, breakfast cereals and multivitamin. After spiking nicotinamide-d4 as an internal standard, infant formula and breakfast cereal samples were hydrolysed under alkaline condition. Samples were then analysed in SRM mode to detect nicotinic acid and nicotinic acid-d4 at m/z 124→80 and 127→84, respectively. In the case of multivitamin sample that contains mainly free nicotinamide, LC/MS monitored nicotinamide and nicotinamide-d4 at their SRM channels of m/z 123→80 and m/z 127→84, respectively, after simple extraction. The repeatability and reproducibility were tested for the validation of the developed ID/LC–MS method. Additionally, the developed analytical method was applied to determine total niacin contents in homogenised infant formula, homogenised multivitamin, and commercially available products including different types of infant formula, breakfast cereals, and multivitamin tablets.

The determination of water‐soluble vitamins and in vitro digestibility of selected Czech cheeses

SummaryWater‐soluble vitamins, in vitro organic matter digestibility (OMD) and dry matter digestibility (DMD) values were determined in various different Czech cheeses. An HPLC method for the simultaneous determination of niacin, pantothenic acid and pyridoxine in cheese was established. Different hydrolysis conditions and enzymes were used to release these vitamins from their complex form. The cheeses contained on average 2.04 mg of niacin, 3.24 mg of pantothenic acid and 2.16 mg of pyridoxine per 100 g of fresh mass. An enzymatic method was applied for the evaluation of OMD and DMD in cheeses. In vitro digestibility values ranged from 83.29% to 99.99% for organic matter (OMD) and 83.33% to 99.87% for dry matter (DMD) after two different enzymatic digestions (pepsin, pancreatin). In addition, cheeses incubated with pepsin plus pancreatin, OMD and DMD were strongly digested (99.04–99.83%, 97.73–99.74%).

Simultaneous determination of niacin and its metabolites—nicotinamide, nicotinuric acid and N‐methyl‐2‐pyridone‐5‐carboxamide—in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

An LC-MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N-methyl-2-pyridone-5-carboxamide (2-Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid-liquid extraction. The chromatographic separation of NA, NAM, NUA, 2-Pyr and IS was achieved on a Hypersil-BDS column (150 x 4.6 mm, 5 microm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2-Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 100-20000 ng/mL for NA; 10-1600 ng/mL for NUA and NAM and 50-5000 ng/mL for 2-Pyr with mean correlation coefficient of ≥ 0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans.

LC-MS–MS Simultaneous Determination of Niacin, Niacinamide and Nicotinuric Acid in Human Plasma LC-MS–MS and Its Application to a Human Pharmacokinetic Study

A highly selective and sensitive liquid chromatographic tandem mass spectrometric (LC-MS–MS) method was developed and validated for the quantitation and pharmacokinetic study of niacin (NA) and its two metabolites niacinamide (NAM) and nicotinuric acid (NUR) in human plasma. Protein precipitation with 14% perchloric acid solution was selected for sample preparation, and ganciclovir was used as an internal standard. Separation was on a Phenomenex Curosil-PFP (250 mm × 4.6 mm, 5 μm) column by a multiple steep steps linear gradient elution with mobile phase consisting of water and methanol, both containing 0.1% formic acid, pumped at a flow rate of 1 mL min−1. The determination was optimized and carried out with positive electrospray ionization by selective multiple reaction monitoring. The method was linear in the concentration range of 15–2,000 ng mL−1 for NA, 70–2,000 ng mL−1 for NAM and 10–2,000 ng mL−1 for NUR, by standard addition calibration. The application of LC-MS–MS was demonstrated for the specific and quantitative analysis of NA, NAM and NUR in human plasma from a pharmacokinetic study in 12 healthy Chinese volunteers treated with three incremental doses of niacin extended-release/lovastatin tablets and an additional steady-state regime.

Development and validation of HPTLC method for niacin and simvastatin in binary combination

A simple, sensitive and validated HPTLC method has been developed to determine Niacin and simvastatin simultaneously in synthetic mixture form. Chromatographic separation was achieved on a RP18 plate using a mixture of Methanol: Water: Acetic acid (60:40:0.1) at a wavelength of 237 nm. Linearity of themethod was found to be in the concentration range of 5000.0-25000.0 μg/ml for niacin and 100.0-500.0 μg/ml for simvastatin with correlation coefficient greater than 0.999. The method can be used for simultaneous determination of Niacin and Simvastatin.

고체상 정제 및 HPLC/PDA에 의한 영유아식 중 나이아신의 분석

This study was performed to establish a rapid and simple analytical method for niacin (nicotinic acid and nicotinamide) using HPLC. A pretreatment method for the extraction and clean-up of niacin in infant formula sample and an instrumental condition for HPLC were optimized. Niacin was extracted by 5 mM hexanesulfonate with ultrasonication for 30 min. For the clean-up of the sample, the extract was applied to a HLB cartridge, and then niacin was eluted from the cartridge using 5 mL of 80% methanol after washing with 5 mL of n-hexane. The total recoveries were 83～104% and relative standard deviation were in the range of 1.5～3.5% during the extraction and clean-up process. Niacin was determined by gradient elution with sodium hexanesulfonate/methanol buffer as a mobile phase and a photodiode array detector (260 nm). It showed a high linearity between the content of niacin and the peak area (r 2 =1.000) in the range of 0.02～10.0 mg/L of nicotinic acid and nicotinamide. The detection limit was 0.02 mg/L (0.2 mg/kg in the sample). The method was successfully applied for the determination of niacin in infant formula. Total niacin contents were in the range of 53.5～140.3 mg/kg.

Assessment of matrix effects and determination of niacin in human plasma using liquid–liquid extraction and liquid chromatography–tandem mass spectrometry

A simple, sensitive and rapid liquid-liquid extraction method for the analysis of nicotinic acid (niacin) and its labeled internal standard nicotinic acid-d4 (niacin-d4) in human plasma was developed and validated. The analyte and its internal standard were isolated from acidified plasma using a single liquid-liquid extraction procedure with methyl-t-butyl ether. The extracted samples were analyzed by liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring. The calibration curves were linear in the measured range between 5 and 1000 ng/mL and the limit of detection was calculated as 122 pg/mL. The method required 250 microL of human plasma and the total run time between injections was 3.5 min. Matrix effects were assessed by post-column infusion experiments, phospholipids monitoring and post-extraction addition experiments. The extraction of phospholipids and niacin from plasma was studied under acidic, neutral and basic conditions. Acidic conditions were optimal for both the recovery of niacin and the removal of phospholipids; the degree of matrix effects for niacin was determined to be 2.5%. It was concluded that effective removal of matrix components can overcome low recovery issues associated with liquid-liquid extractions of polar analytes.

Determination of Niacin in Food Materials by Liquid Chromatography Using Isotope Dilution Mass Spectrometry

The availability of deuterium-labeled nicotinic acid makes stable isotope dilution mass spectromerty (MS) coupled with liquid chromatography (LC) an attractive option for the determination of the water-soluble B-vitamin niacin in food samples. A method was developed based on acid digestion, solid-phase extraction with a strong cation exchange column, and reversed-phase chromatography with a C18 column. Detection is by positive ion electrospray MS. Analysis in selected ion recording mode is subject to interference problems similar to those found with other LC determinations of niacin, but the additional selectivity of multiple reaction monitoring mode largely eliminates interference problems. The method was applied to 6 different food matrixes and to appropriate reference materials, including milk samples with niacin levels near 1 ppm. The method exhibited good accuracy, based on levels obtained for the reference materials, and relative standard deviations in the range of 0.5-5%.

Solid Phase Extraction/Liquid Chromatography Method for the Determination of Niacin in Commercial Flour Products

Solid Phase Extraction/Liquid Chromatography Method for the Determination of Niacin in Commercial Flour Products

Determination of niacin in fresh and dry cured pork products by ion chromatography: experimental design approach for the optimisation of nicotinic acid separation

A simple and robust method to determine the niacin level in fresh and dry-cured pork products by ion chromatography is reported. The analytical procedure includes an acidic hydrolysis to free all the bound forms (NAD and NADP) and to convert all the niacin into nicotinic acid, after that the chromatographic separation of the acid form is performed by a cation-exchange column and UV detection (262 nm). The evidence of several interferences that did not allow a good resolution of the nicotinic acid peak in fresh meat matrix led us to improve the resolution by means of statistical optimisation. A three level factorial design and response surface methodology were applied to optimise the chromatographic conditions (mobile phases compositions) and to assess the robustness of the method. Analytical performances of the optimised method were investigated in terms of linearity, limit of detection (LOD) and precision. The complete analytical procedure to extract and determine the nicotinic acid in meat was validated by IC-MS.

Simultaneous determination of niacin, niacinamide and nicotinuric acid in human plasma

A sensitive, specific, accurate, and reproducible HPLC/MS-method for the simultaneous quantitative determination of niacin (NA) and its main metabolites niacinamide (NAM) and nicotinuric acid (NUR) in human plasma using chinolin-3-carboxylic acid as an internal standard was developed and validated according to international guidelines for method validation. All analytes and the internal standard were separated from acidified plasma by solid phase extraction. Afterwards the extracted samples were analyzed by HPLC/MS in the positive electrospray ionization mode (ESI) and selected ion monitoring (SIM). The total run time was 7 min between injections. The assay had a lower limit of quantification of 50.0 ng/mL for each analyte using 1 mL of plasma. The calibration curves were linear in the measured range between 50.0 and 750 ng/mL plasma. The overall precision and accuracy for all concentrations of quality controls and standards was better than 15%. No indications were found for possible instabilities of niacin, niacinamide and nicotinuric acid in plasma at −20 °C, in the extraction solvent or after repeated thawing/freezing cycles. In stabilities were observed in whole blood and in plasma at room temperature. The recovery of the extraction method ranged from 86 to 89% for the three analytes.

Determination of water-soluble vitamins in soft drinks and vitamin supplements using capillary electrophoresis.

A method for the determination of six water-soluble vitamins based on capillary electrophoresis (CE) operated in micellar mode was developed. Thiamine hydrochloride (vitamin B1), riboflavin (vitamin B2), pyridoxine hydrochloride (vitamin B6), pantothenic acid (vitamin B5), nicotinamide (vitamin B3), and cobalamin (Vitamin B12) could be separated in a single run. All CE parameters such as buffer composition and operation temperature were optimized in order to achieve better separation. Relative standard deviations (RSDs) of the described method ranged from 1.08 to 3.68% (intra-day precision) and 1.26 to 3.35% (inter-day precision). The method was then used for measuring various soft drinks and vitamin supplements directly without any step of sample cleanup. The determination of niacin was successful for all samples tested, reaching recoveries near 100%. Riboflavin and pyridoxine were quantified successfully in some but not all samples. Therefore, an evaluation on a case-by-case basis is mandatory. When applicable, this method provides a fast, accurate, simple, and inexpensive way to quantify selected vitamins, and is therefore well suited for routine analysis in soft drink industry.

Asia Pacific food analysis network (APFAN) training exercise: the determination of niacin in cereals by alkaline extraction and high performance liquid chromatography

Analysts from 20 laboratories, mainly from developing countries in the Asia Pacific Food Analysis Network (APFAN), were invited to participate in a training exercise for the determination of niacin (vitamin B 3) in cereals using alkaline extraction and high performance liquid chromatography (HPLC) as the determinative step. The purpose of the study was to assist these laboratories in adopting a robust HPLC method for the determination of niacin in these foods. The exercise was divided into three discrete phases. Analysts were supplied with the validated method together with all of the chemicals and solid-phase extraction columns that were necessary to complete the three phases of the exercise. Phase 1 of the study consisted of a “hands on” workshop for 10 overseas analysts conducted at the 6th APFAN workshop held at the Queensland Health Scientific Services Laboratory, Brisbane, Australia, in May 1999. In phase 1, each analyst was requested to determine the niacin content of three samples of finely ground breakfast cereal that contained a known amount of niacin. Phase 2 involved these analysts as well as analysts from 12 other laboratories in the APFAN network. The analysts were requested to determine the niacin content of a fortified reference material (American Association of Cereal Chemists VMA 195) containing niacin in the range 13.9–20.1 mg/100 g in their own laboratories. Twelve participants reported levels of niacin ranging from 10.6 to 21.7 mg/100 g. Analysts who successfully completed this phase then participated in phase 3, involving the analysis of five different cereal types of unknown niacin content. Eight sets of data were reported for phase 3. Approximately half of the data were in the expected range for the levels of niacin in the samples.

Enzymatic extraction procedure for the liquid chromatographic determination of niacin in foodstuffs

An enzymatic extraction procedure for the determination of the niacin present in foodstuffs by means of a NAD glycohydrolase (or NADase) has been proposed as a replacement for the hydrochloric acid hydrolysis usually used. The determination of nicotinic acid and nicotinamide by HPLC/fluorimetry [according to Lahély et al. (Food Chemistry, 1999, 65, 129)] has made it possible to show that the acid treatment very definitely led to the release of forms of nicotinic acid not bioavailable in some foodstuffs (wheat flour, wheat germ, peanuts), unlike hydrolysis with NADase from Neurospora crassa [37 °C, 18 h, pH 4.5, 24 μg g−1 of sample (0.013 U g−1)] which led to both an accurate determination of the respective contents of nicotinic acid and nicotinamide and a reliable estimation of the bioavailable niacin in the different foodstuffs analysed. The combined addition of a protease and an amylase to the NADase, sometimes recommended when acid hydrolysis was used, proved to be superfluous.

Determination of Niacin in Infant Formula by Solid-Phase Extraction/Liquid Chromatography: Peer-Verified Method Performance–Interlaboratory Validation

This paper reports the results of the interlaboratory peer validation study of AOAC Peer-Verified Method (PVM) 1:2,000 for the determination of niacin in infant formula by solid-phase extraction/liquid chromatography. We have used a Data Quality Objectives (DQO) approach to address not only method variability and robustness but also accuracy of data through the use of an appropriate reference material in conjunction with the interlaboratory validation study. Our DQO included the following: (1) statistical agreement of analytical results and quantitative recovery between 2 collaborating laboratories; (2) the repeatability relative standard deviation (RSDr) values and the HORRAT (Horwitz ratio) obtained (1.07), which satisfied the criteria of the Horwitz "limits of acceptability" at the analyte level present; (3) validation of lack of interference; and (4) accuracy agreement within assigned values for a certified reference material. National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1846 Infant Formula, with a certified value of 63.3 +/- 7.6 microg/g for niacin content, was used as a test material for collaborative study and accuracy assessment. Niacin values obtained by the originating laboratory were 59.7 +/- 4.0 microg/g (95% confidence interval [CI] = 1.4 microg/g with a relative standard deviation [RSD] of 6.7%) and by the peer laboratory were 56.6 +/- 6.6 microg/g (95% CI = 4.1 microg/g, with an RSD of 11.7%). Statistical evaluation using the means equivalence test showed that nicotinic acid values obtained by the peer laboratory were equivalent to those values obtained by the originating laboratory. Linear calibration curves and quantitative recovery were obtained. Integration of the PVM process with a readily available certified reference material gives the user confidence in the accuracy of the data generated by the method through traceability to the reference material used.

EVALUATION OF NIACIN LC METHODS BY DIODE ARRAY/SPECTRAL ANALYSIS

ABSTRACT Three extraction/LC methods for the determination of niacin in food materials have been qualitatively evaluated by spectral analysis procedures using a photodiode array detection system. This system allows visual inspection of multiple wavelength scans to identify presence of multiple components under LC peaks. It also allows calculation of a Similarity Index (SI) by means of library match software that compares the spectra of a LC peak to that of a standard sample. Niacin can occur in foods either as nicotinic acid or often as added nicotinamide to enrich foods. We have evaluated LC methods for determination of both forms of niacin in several reference materials: SRM 1846 Infant Formula (NIST), RM 8437 Hard Red Spring Wheat (NIST), and VMA 195 Cereal (AACC). Spectral analysis showed only the niacin vitamer with a library match having a SI>0.99 (1.0 is a perfect match) for nicotinic acid by anion exchange LC using an aromatic sulfonic acid–cation exchange solid phase extraction following a standard H2SO4 digest, and for nicotinamide using a trichloroacetic acid (TCA) extraction followed by reverse phase (RP) LC. A method involving meta-phosphoric acid (m-PA) extraction followed by RP/LC is not as satisfactory; while the 3-D view is not conclusive for a multiple component peak, the library match indicates a significantly lower SI of 0.81. Utilization of spectral analysis software with library matching is a useful tool for evaluating a variety of LC protocols. More robust LC methods for determining niacin in foods can replace laborious microbiological methods currently used by food testing laboratories. These LC methods combined with use of appropriate reference materials can provide definitive data for niacin in foods to be incorporated in food compositional tables, for use in food labeling and by dieticians preparing formulated diets.