液相色谱法测定多元维生素片中4种水溶性维生素

采用高效液相色谱法测定多元维生素片中盐酸硫胺(B1)、核黄素(B2)、泛酸钙(B5)、盐酸吡哆醇(B6)的含量。重点考察了提取条件对于维生素测定的影响,4种维生素在确定的浓度范围内分别与色谱峰面积呈良好的线性关系,r2≥0.9999,加标回收率为99.2%～101.4%。4种维生素的检出限分别为0.30、0.19、0.45、0.11μg/g(S/N10)。该方法可作为实际测试标准方法使用。     

Determination of water-soluble vitamins by liquid chromatography with a parallel dual-electrode electrochemical detector

A method for the determination of water-soluble vitamins (ascorbic acid, pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxamine dihydrochloride, p-aminobenzoic acid, folic acid) by liquid chromatography, with a parallel dual-electrode electrochemical detector, is described. One electrode was controlled at +0.80 V (vs. SCE), the other at +1.20 V (vs. SCE). The possibility of interference by eight other water-soluble vitamins (riboflavin, nicotinamide, cyanocobalamin, menadione, dextro calcium pantothenate, thiamine, nicotinic acid, dextro biotin) was studied. These vitamins did not interfere when a parallel dual-electrode detector system was used. The estimation of five of the vitamins was studied in detail. The linear ranges found were 10 ng-1.2 mug for pyridoxine hydrochloride, 2 ng-2mug for pyridoxal hydrochloride, 10 ng-3 mug for pyridoxamine dihydrochloride, 5-200 ng for folic acid and 0.6-200 ng for p-aminobenzoic acid, the limits of detection being 3, 0.6, 1, 2 and 0.06 ng respectively. Application of the technique to the estimation of vitamin B(6) in tablets is illustrated. The results indicate that the vitamin B(6) in these tablets existed in the pyridoxine hydrochloride form and the B(6) content agreed well with liquid chromatograph by spectrophotometric analysis.     

Multisyringe Chromatography (MSC): An Effective and Low Cost Tool for Water-Soluble Vitamin Separation

The advent of monolithic columns has facilitated the combination of chromatographic techniques with nonseparation flow techniques by virtue of the low overpressure introduced by them. Thus far, these combinations have provided excellent results derived from the high selectivity of liquid chromatography and ease of sample handling in nonseparation flow techniques. The joint use of multisyringe flow injection analysis and monolithic columns has provided multisyringe chromatography (MSC).

Simultaneous spectrophotometric determination of ascorbic acid, nicotinic acid, nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, and riboflavin in commercial drinks using a MSC method, is described in this contribution as an inexpensive alternative to classical HPLC. The MSC method provides baseline separation of all these compounds with resolutions values greater than 2, except for B1 and B6 with resolution of 1,2.

A ChromolithTM Flash RP-18e (50 mm × 4.6 mm) column, a multisyringe burette provided with two 10 mL high-precision bidirectional syringes and an 8-port multiposition valve were used for a dual isocratic chromatographic separation in our study.     

Simultaneous determination of water-soluble vitamins in a vitamin-enriched drink by an in-capillary enzyme reaction method

The in-capillary enzyme reaction method was used to determine riboflavin phosphate in a vitamin-enriched drink based on its conversion to riboflavin (vitamin B2) with alkaline phosphatase. Simultaneously, three water-soluble vitamins [thiamine nitrate (vitamin B2 mononitrate), pyridoxine hydrochloride (vitamin B6 hydrochloride) and nicotinamide (vitamin PP)] and anhydrous caffeine in the drink were subjected to quantitative analysis. In the system, electrophoretic migration was used to mix zones containing the substrate (riboflavin phosphate) and the enzyme (alkaline phosphatase). The reaction was then allowed to proceed in the presence of a weak electric field and, finally, the product (riboflavin) of enzyme reaction and other water-soluble vitamins migrated under the influence of an applied electric field to the detector. All the active ingredients and the formulation excipients were successfully separated by micellar electrokinetic chromatography with 135 mM sodium dodecyl sulfate. To prevent inhibition of enzyme reaction by the addition of sodium dodecyl sulfate to the reaction zone, sandwich mode injection, in which plugs of sandwich solution without sodium dodecyl sulfate were introduced into the capillary on both sides of the reaction zone, was utilized as a barrier to protect the enzyme reaction from the inhibitor. The relationship between the peak area of the product and the concentration of the substrate was calculated in the in-capillary enzyme reaction method. Excellent linearity was obtained, with correlation coefficients of 0.9999. The established method was validated and demonstrated to be applicable to the determination of the five active ingredients, including riboflavin phosphate, in a commercial vitamin-enriched drink. No interference from the formulation excipients was observed. Good linearities were obtained, with correlation coefficients above 0.999. Recoveries and precisions ranged from 99.3 to 101.8%, and from 0.1 to 2.5% RSD, respectively. Good agreement was obtained between the established method and traditional high-performance liquid chromatographic methods. These results suggest that the in-capillary enzyme reaction method can be used for the simultaneous determination of riboflavin phosphate and other water-soluble vitamins in pharmaceuticals.     

Development of a validated HPLC method for the determination of B-complex vitamins in pharmaceuticals and biological fluids after solid phase extraction

An HPLC method was developed for the simultaneous determination of seven water-soluble vitamins, viz. thiamine, riboflavin, nicotinic acid, nicotinamide, pyridoxine, cyanocobalamin, and folic acid, in multivitamin pharmaceutical formulations and biological fluids (blood serum and urine). Separation was achieved at ambient temperature on a Phenomenex Luna C18 (150 x 4.6 mm) analytical column. Gradient elution was performed starting at a 99:1 A:B v/v composition, where A: 0.05 M CH3COONH4/CH3OH (99/1) and B: H2O/CH3OH (50/50), at a flow rate of 0.8 mL/min. After a 4-min isocratic elution the composition was changed to 100% of B in 18 min and elution continued isocratically for 8 min. Detection was performed with a photodiode array detector at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples. Detection limits were in the range of 1.6-3.4 ng, per 20-microL injection, while linearity held up to 25 ng/microL. Accuracy, intra-day repeatability (n = 6), and inter-day precision (n = 7) were found to be satisfactory. Theobromine (2 ng/microL) was used as internal standard. Sample preparation of biological fluids was performed by SPE on Supelclean LC-18 cartridges with methanol-water 85/15 v/v as eluent. Extraction recoveries from biological matrices ranged from 84.6% to 103.0%.     

Simultaneous determination of some water-soluble vitamins and preservatives in multivitamin syrup by validated stability-indicating high-performance liquid chromatography method

HPLC stability-indicating method has been developed for the simultaneous determination of some water-soluble vitamins (ascorbic acid, thiamine hydrochloride, riboflavin-5'-phosphate sodium, pyridoxine hydrochloride, nicotinamide, D(+)-panthenol) and two preservatives (methylparaben and sodium benzoate) in multivitamin syrup preparation. Water-soluble vitamins, preservatives and their degradants were separated on Zorbax SB-Aq (C(18)) (250 mm x 4.6 mm, 5 microm) column at an ambient temperature. Combined isocratic and gradient elution was performed with a mobile phase consisting of 0.0125 M hexane-1-sulfonic acid sodium salt in 0.1% (m/v) o-phosphoric acid, pH 2.4-2.5 (solvent A) and acetonitrile (solvent B) at the flow-rate 1 ml min(-1). Starting with solvent A an isocratic elution was performed for 15 min, then the composition was changed to 85% of A and 15% of B during the next 20 min and it was constant for 5 min, then the composition was changed to 70% of A and 30% of B during next 15 min and it was constant for 5 min and finally was changed to 100% of A as at the beginning of the elution. Detection was performed with diode array detector at 210, 230 and 254 nm. Multivitamin syrup preparation was subjected to stress testing (forced degradation) in order to demonstrate that degradants from the vitamins, preservatives and/or product excipients do not interfere with the quantification of vitamins and preservatives. Typical validation characteristics: selectivity, accuracy, precision, linearity, range, limit of quantification and limit of detection were evaluated for vitamins and preservatives.     

Simultaneous determination of a ternary mixture of doxylamine succinate, pyridoxine hydrochloride, and folic acid by the ratio spectra-zero-crossing, double divisor-ratio spectra derivative, and column high-performance liquid chromatographic methods

Three simple, rapid, and accurate methods, i.e., the derivative ratio spectra-zero-crossing method (method I), double divisor-ratio spectra derivative method (method II), and column reversed-phase high-performance liquid chromatographic (RP-HPLC) method (method III) were developed for the simultaneous determination of doxylamine succinate (DOX), pyridoxine hydrochloride (PYR), and folic acid (FA) in their ternary mixtures and in tablets. In methods I and II, the calibration graphs were linear in the range of 2.5-80, 1.0-40, and 1.0-30 microg/mL for DOX, PYR, and FA, respectively. In the HPLC method, the separation of these compounds was performed using mobile phase consisting of 0.05 M phosphate buffer (pH 6.3)-methanol-acetonitrile (50 + 20 + 30, v/v/v), and UV detection was performed at 263 nm. Linearity was observed between the concentrations of the analytes and peak areas [correlation coefficient (r) > or =0.9998] in the concentration range of 1.0-200, 4.0-600, and 4.0-600 microg/mL for DOX, PYR, and FA, respectively. The standard deviation of retention time in method III was 0.011, 0.015, and 0.016 for DOX, PYR, and FA, respectively. The precision studies for all of the methods gave relative standard deviation values of <2%. The results obtained from the methods were statistically compared by means of Student's t-test and the variance ratio F-test. It was concluded that all of the developed methods were equally accurate, sensitive, and precise. These methods could be applied to determine DOX, PYR, and FA in their combined dosage forms.     

Determination of eight water- and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by high-performance liquid chromatography

In the present work, a reversed-phase high-performance liquid chromatographic procedure has been developed for the determination of water-soluble vitamins (thiamine hydrochloride, pyridoxine hydrochloride, nicotinamide, riboflavin phosphoric ester and cyanocobalamine) and fat-soluble vitamins (retinol palmitate, cholecalciferol, -tocopherol acetate) in multi-vitamin pharmaceutical formulations. The sample treatment proposed consists of a solid-phase extraction with C₁₈ AR cartridges that allow the separation of fat-soluble vitamins, which were retained on the sorbent, from water-soluble vitamins. Afterwards, the water-soluble vitamins were analysed by HPLC on a Nova-Pack C₁₈ (150×3.9 mm, 4 μm) analytical column, using CH₃OH–0.05 M CH₃COONH₄ as mobile phase The chromatographic analysis of the fat-soluble vitamins was carried out after their sequential elution with methanol and chloroform from C₁₈ sorbent, on the above column. The mobile phase employed was MeOH–CH₃CN (95:5, v/v) working at a flow-rate of 2 ml min⁻¹ in isocratic mode. The solid-phase extraction for these vitamins had been previously optimised. The experimental variables studied were: application volume, elution solvents and cleaning solutions. The UV–Vis detection of vitamins was made at 270 nm for all the water-soluble vitamins (362 nm for B₁₂) and 285 nm for the water-soluble and fat-soluble vitamins present in real samples at different concentration levels. The accuracy of the method was tested obtaining an average recovery ranging between 78 and 116%.     

Electrochemical behaviour of pyridoxine hydrochloride (vitamin B6) at carbon paste electrode modified with crown ethers

The electrochemical behaviour of pyridoxine hydrochloride (pyridoxine HCl) at the plain carbon paste electrode and the electrode modified with oxa crown ether has been studied using voltammetric and impedance measurements. The macrocycles used as modifiers were 18-crown-6, dibenzo-18-crown-6 (DB18C6), dicyclohexano-18-crown-6 and dibenzo-24-crown-8, out of which DB18C6 gave better response for pyridoxine HCl. Tris buffer (pH 10.3) was chosen as an appropriate medium among the several supporting electrolytes of varying pH studied. The characterization of the DB18C6-modified electrode (CME-DB18C6) using kinetic parameters such as number of electrons (n) and electron transfer coefficient (α) is studied by cyclic voltammetry. Electrochemical impedance spectroscopic measurements obtained confirm the current enhancement over the modified electrode. Analytical applications of this electrode have been studied for the determination of pyridoxine HCl. A sensitive linear working range of 0.6 to 100 μg cm⁻³ with a detection limit of 0.4 μg cm⁻³ by differential pulse voltammetry was observed for pyridoxine HCl on CME-DB18C6. However, on decreasing the scan rate to 5 mV s⁻¹, the detection limit lowered to 0.2 μg cm⁻³. Interference from some vitamins like thiamine hydrochloride, riboflavin, nicotinamide, para-aminobenzoic acid, cyanocobalamin, folic acid and d-biotin and amino acid l-tryptophan was studied, and simultaneously, riboflavin, thiamine hydrochloride and pyridoxine HCl were determined over the modified electrode, CME-DB18C6. The modified electrode is successfully used for the determination of pyridoxine HCl in multivitamin pharmaceutical preparations.     

Ion‐pair cloud‐point extraction: A new method for the determination of water‐soluble vitamins in plasma and urine

A novel, simple, and effective ion‐pair cloud‐point extraction coupled with a gradient high‐performance liquid chromatography method was developed for determination of thiamine (vitamin B₁), niacinamide (vitamin B₃), pyridoxine (vitamin B₆), and riboflavin (vitamin B₂) in plasma and urine samples. The extraction and separation of vitamins were achieved based on an ion‐pair formation approach between these ionizable analytes and 1‐heptanesulfonic acid sodium salt as an ion‐pairing agent. Influential variables on the ion‐pair cloud‐point extraction efficiency, such as the ion‐pairing agent concentration, ionic strength, pH, volume of Triton X‐100, extraction temperature, and incubation time have been fully evaluated and optimized. Water‐soluble vitamins were successfully extracted by 1‐heptanesulfonic acid sodium salt (0.2% w/v) as ion‐pairing agent with Triton X‐100 (4% w/v) as surfactant phase at 50°C for 10 min. The calibration curves showed good linearity (r² > 0.9916) and precision in the concentration ranges of 1‐50 μg/mL for thiamine and niacinamide, 5–100 μg/mL for pyridoxine, and 0.5–20 μg/mL for riboflavin. The recoveries were in the range of 78.0–88.0% with relative standard deviations ranging from 6.2 to 8.2%.     

Nanocrystalline Metallosilicate Modified Electrodes for the Simultaneous,Sensitive, and Selective Determination of Riboflavin,Rutin, and Pyridoxine

Nanocrystalline metallosilicate modified glassy carbon electrodes were fabricated for the simultaneous determination of vitamins. Among these, nanocrystalline zirconosilicate exhibited the highest activity with a linear range from 30 nM–500 µM for riboflavin and 120 nM–600 µM for rutin and pyridoxine. Sensitivity values of 2.8, 1.49, and 1.13 µA/µM cm² and lower detection limits of 5 nM, 30 nM, and 30 nM for riboflavin, rutin, and pyridoxine, respectively, were found. The proposed sensor is stable and reproducible (RSD<3.5 %). The analytical performance of this sensor was demonstrated in the pharmaceutical preparations (multivitamin tablets) with satisfactory recovery (97–103 %).     

High-performance column liquid chromatographic method for the simultaneous determination of buclizine, tryptophan, pyridoxine, and cyanocobalamin in tablets and oral suspension

An HPLC method was developed and validated for the simultaneous determination of buclizine, tryptophan, pyridoxine, and cyanocobalamin in pharmaceutical formulations. The chromatographic separation was carried out on an RP-C18 column using a mobile phase gradient of methanol, 0.015 M phosphate buffer (pH 3.0), and 0.03 M phosphoric acid at a flow rate of 1.0 mL/min and UV detection at 230, 280, and 360 nm, respectively, for buclizine, tryptophan, pyridoxine, and cyanocobalamin. The method validation yielded good results with respect to linearity (r>0.999), specificity, precision, accuracy, and robustness. The RSD values for intraday and interday precision were below 1.82 and 0.63%, respectively, and recoveries ranged from 98.11 to 101.95%. The method was successfully applied for the QC analysis of buclizine, tryptophan, pyridoxine, and cyanocobalamin in tablets and oral suspension.     

A porous graphitized carbon column HPLC method for the quantification of paracetamol, pseudoephedrine, and chlorpheniramine in a pharmaceutical formulation

A simple, rapid, and stability-indicating HPLC method has been developed, fully validated, and applied to the quantification of paracetamol, pseudoephedrine hydrochloride, and chlorpheniramine maleate in a pharmaceutical formulation, using hydrochlorothiazide as an internal standard. Chromatographic separation was achieved isocratically on an RP porous graphitized carbon analytical column (125 x 2.1 mm id, particle size 5 microm) using 5.0 mM ammonium acetate-acetonitrile (35 + 65, v/v) mobile phase at a flow rate of 0.50 mL/min. UV spectrophotometric detection at 220 nm was used. The method had linear calibration curves over the range of 30-70 microg/mL for paracetamol, 1.8-4.2 microg/mL for pseudoephedrine hydrochloride, and 120-280 ng/mL for chlorpheniramine maleate. The intraday and interday RSD values were less than 3.2% for all compounds, while the relative error was less than 2.9%. Accelerated stability studies performed under various stress conditions proved the selectivity of the method. The developed method was applied successfully to QC and content uniformity tests of commercial tablets.     

Simultaneous determination of riboflavin, pyridoxine, nicotinamide, caffeine and taurine in energy drinks by planar chromatography-multiple detection with confirmation by electrospray ionization mass spectrometry

A new high-throughput method was developed to detect simultaneously riboflavin (Vitamin B(2)), pyridoxine (Vitamin B(6)), nicotinamide (Vitamin B(3)), caffeine and taurine in energy drinks by multiple detection. Ten samples of energy drinks and six samples of beverages containing caffeine were prepared by degassing in an ultrasonic bath for 20 min. After chromatography, multi-wavelength scanning was performed by: (1) UV-absorbance measurement at 261 nm for nicotinamide and 275 nm for caffeine, (2) fluorescence measurement at 366/>400 and 313/>340 nm for riboflavin and pyridoxine, respectively, and (3) Vis-absorbance measurement at 525 nm for taurine, after post-chromatographic derivatization with ninhydrin reagent. Calibrations were linear or polynomial with determination coefficients r(2)>0.999. Overall recoveries of the five compounds were between 81 and 106% at three different concentration levels. Repeatabilities (RSD, %) of all substances in matrix were determined to be between 0.8 and 1.5%. Intermediate precisions (RSD, %) ranged between 3.6 and 7.4% for riboflavin, 2.8 and 6.3% for nicotinamide, 2.5 and 4.4% for caffeine, 2.1 and 2.9% for taurine and 0.5 and 4.0% for pyridoxine at different concentration levels. Mass confirmation was performed by a single quadrupole MS in positive electrospray ionisation (ESI) scan mode for all substances except taurine (negative mode). This method offers a good alternative for routine analysis due to its simplicity and at the same time reliability.     

Simultaneous determination of thiamine and riboflavin in edible marine seaweeds by high-performance liquid chromatography

This study presents a high-performance liquid chromatography (HPLC) method for simultaneous determination of thiamine and riboflavin and the results of its application to a number of edible seaweeds that are sampled in dried form (Himanthalia elongata, Laminaria ochroleuca, Undaria pinnatifida, Palmaria sp., and Porphyra sp.) or as canned food (H. elongata and Saccorhiza polyschides). Samples are prepared by acid and enzymatic hydrolysis. Optimized conditions for reversed-phase HPLC with fluorescence detection are as follow: column, Kromasil 100 C18; column temperature, 35 degrees C; mobile phase, a 72:28 (v/v) mixture of 0.005 M ammonium acetate (pH 6.7)-methanol; and flow rate, 1.35 mL/min. With these conditions, recovery is 95.52% for thiamine and 90.08% for riboflavin, and the method precision (relative standard deviation) is 2.66% for thiamine and 2.21% for riboflavin. On a dry weight basis, thiamine contents range from 0.14 microg/g in dried H. elongata to 2.02 microg/g in dried Porphyra and riboflavin contents from 0.31 microg/g in canned H. elongata to 6.15 microg/g in dried Porphyra.     

Micellar liquid chromatography determination of B vitamins with direct injection and ultraviolet absorbance detection

A micellar reversed-phase liquid chromatographic procedure was developed for the control of five water-soluble vitamins, B (nicotinamide), B1 (thiamine), B2 (riboflavin), B6 (pyridoxine and pyridoxamine), in multivitamin pharmaceutical formulations (capsules, pills and syrups). Optimization procedure includes studies about the composition of the mobile phase (sodium dodecyl sulphate and the modifiers propanol, butanol or pentanol), flow-rate and temperature. Chromatographic analysis of all vitamins was carried out using a single mobile phase of 0.1 M SDS-4% (v/v) pentanol at pH 3, in a C18 column in isocratic mode, and UV-detection at 270, 290 and 325 nm. The flow-rates selected were 1.0 ml/min in the interval 0 to 6 min, and 2.0 ml/min until the end of the chromatogram and temperature was 45 degrees C. In the micellar liquid chromatographic system, the samples were injected without pretreatment, and the analysis time was below 12 min. Repeatabilities and intermediate precision were achieved according to ICH, and were below 5%. When the method is applied to real samples, the amount found with respect to the declared compositions were within the 91-105% range. These results were similar to those obtained with a conventional 60:40 (v/v) methanol-water mixture for some of the vitamins, but with the advantage of use a single mobile phase for the analyses of the five vitamins, with direct injection of the samples and reduced toxicity, flammability, environmental impact and cost of the micellar-pentanol solutions.     

LC/UV/MS-MRM for the simultaneous determination of water-soluble vitamins in multi-vitamin dietary supplements

The purpose of this study was to optimize chromatographic and detection conditions for the simultaneous determination of water-soluble vitamins in multi-vitamin dietary supplements using a single chromatographic run. An approach using liquid chromatography with diode array and/or mass spectrometry for quantitation of seven B-complex vitamins [thiamine (B₁), riboflavin (B₂), nicotinamide (B₃), pyridoxine (B₆) pyridoxine, biotin, pantothenic acid, and folic acid] in multi-vitamin/multi-mineral daily supplements is described. This approach utilizes a reversed phase C18 column (4 μm; i.d.: 250×2.0 mm) with a gradient mobile elution profile, performed at a flow rate of 0.25 ml/min. After a 5-min isocratic elution at 100% A (0.1% formic acid in water), a linear gradient to 50% A and 50% B (0.1% formic acid in acetonitrile) at 15 min and then to 5% A and 95% B at 17 min was employed. Detection was performed with a photodiode array detector (DAD) in sequence with a triple-quad mass spectrometer in the multiple reaction mode (MS-MRM). Although good chromatographic separation of ascorbic acid was also obtained in extracts from multi-vitamin/multi-mineral supplements, the ascorbic acid could not be quantified properly due to rapid oxidation catalyzed by the minerals. This method was initially applied to determine water-soluble vitamins in representative multi-vitamin/multi-mineral tablets following the extraction of ground samples with a phosphate buffer (10 mM, pH 2.5). For multi-vitamin supplement tablets, this approach does not require any sample clean-up/pre-concentration steps except for centrifugation and filtration of the extract.      

Development and validation of an HPLC method for mefloquine hydrochloride determination in tablet dosage form

A simple HPLC method for determination of mefloquine hydrochloride in tablets was developed and validated. The separation was carried out on an Xterra RP18 (250 x 4.6 mm id, 5 pm particle size) analytical column. The mobile phase was 0.05 M monobasic potassium phosphate buffer (pH 3.5)-methanol (40 + 60, v/v). The flow rate and wavelength were set to 1 mL/min and 283 nm, respectively. The method was specific for mefloquine hydrochloride in the presence of hydrolytic, oxidative, and photolytic degradation products. It was also linear, precise, accurate, and robust, being suitable for routine QC analyses and stability studies. The developed HPLC method was compared to a previously described spectrophotometric method.     

高效液相色谱分离-在线光化学衍生/荧光光谱法测定5种水溶性维生素的研究

建立了高效液相色谱分离-在线光化学衍生/荧光光谱法测定水溶性维生素烟酸(NIA)、烟酸胺(NIC)、B1、B12及B2的新方法.以含有0.018 mol/L三乙胺、0.002 mol/L庚烷磺酸钠的0.05 mol/L磷酸盐缓冲液(A相,pH 5.8)和甲醇(B相)为流动相(85:15),等度洗脱分离5种水溶性维生素;...     

Assay of vitamin B in urine by capillary electrochromatography with methacrylate-based monolithic column

A novel and simple method for the separation of major vitamin B analytes, such as thiamine, riboflavin, nicotinamide, vitamin B4, pyridoxine, has been developed by CEC using the monolithic column. It has been found that the baseline separation of the five analytes could be achieved with 5.0 mM phosphate buffer at pH 4.0. Compared with the open-tubular capillary and the bared capillary columns, the poly(butylmethacrylate-co-ethylene glycol dimethacrylate) monolithic capillary could exhibit the best resolution in the analysis. Then the method was validated and the linear calibration ranges were obtained with correlation coefficients more than 0.997. The precision and the recovery were also investigated and showed a good result. Furthermore, the proposed method was successfully applied to assay the concentration of vitamin B analytes and the metabolic situation in human urine samples.