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%.     

Rapid determination of water- and fat-soluble vitamins with microemulsion electrokinetic chromatography

A rapid, reliable and reproducible method based on microemulsion electrokinetic chromatography (MEEKC) for simultaneous determination of 13 kinds of water- and fat-soluble vitamins has been developed in this work. A novel microemulsion system consisting of 1.2% (w/w) sodium lauryl sulphate (SDS), 21% (v/v) 1-butanol, 18% (v/v) acetonitrile, 0.8% (w/w) n-hexane, 20mM borax buffer (pH 8.7) was applied to improve selectivity and efficiency, as well as shorten analysis time. The composition of microemulsion used as the MEEKC running buffer was investigated thoroughly to obtain stable separation medium, as well as the optimum determination conditions. Acetonitrile as the organic solvent modifier, pH of the running buffer and 1-butanol as the co-surfactant played the most important roles for the separation of the fat-soluble vitamins, water-soluble vitamins and stabilization of system, respectively. The 13 water- and fat-soluble vitamins were baseline separated within 30 min. The system was applied to determine water- and fat-soluble vitamins in commercial multivitamin pharmaceutical formulation, good accuracy and precision were obtained with recoveries between 97% and 105%, relative standard derivations (RSDs) less than 1.8% except vitamin C, and acceptable quantitative results corresponding to label claim.     

Determination of water-soluble vitamins in vitamin premixes,bioacitve dietary supplements,and pharmaceutical preparations using high-efficiency liquid chromatography with gradient elution

The influence of the pH and temperature of the mobile phase on the retention and separation of 14 vitamins and vitameric forms has been studied for four different stationary phases. The chromatographic conditions allowing the separation of 12 water-soluble vitamins and vitameric forms have been proposed. It has been established that the best separation of water-soluble vitamins can be achieved by employing gradient elution. The mobile phase A, (0.6% phosphoric acid) pH 1.5–1.8; mobile phase B, acetonitrile. For the separation of nicotinic and ascorbic acid it is preferable to use stationary phases Luna C18(2) and Gemini C18, while for the separation of riboflavin and riboflavin-5-phosphate the Synergy Hydro RP and Zorbaz SB-C18 stationary phases should be used. The selected conditions have been used for the determination of vitamins in commercial samples of vitamin preparations. The results are in good accordance with the producer data.     

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.     

A liquid chromatography method for determination of selected amino acids, coenzymes, growth regulators, and vitamins from Cicer arietinum (L.) and Solanum lycopersicum (L.)

A bottleneck in crosstalk and QC research has been the quantification of diverse chemotypes in small amounts of tissue. An LC-UV method for estimating 28 selected metabolites of the regulatory network underlying growth, development, maintenance, vital functions, defense reactions, and food quality is reported. The method was based on binary gradient resolutions of the analytes in an RP C18 column. The mobile phase comprised solvent A [water+0.1% trifluoroacetic acid (TFA)] and B (acetonitrile + 0.085% TFA at a flow rate of 1 ml/min. Twenty-three metabolites (selected amino acids, coenzymes, growth regulators, phenolic antioxidant, and water-soluble vitamins) were detected at 254 nm, and four fat-soluble vitamins at 280 nm. Jasmonic acid was quantified at 210 nm. The RSDs of peak area and retention time for each metabolite were <5.8%. The calibration graphs were linear with R2 values ranging from 0.98 to 0.99. The LODs (microg/mL) were about 0.01-1.0 for 23 metabolites quantified at 254 nm, 0.1-0.2 for fat-soluble vitamins, and 0.1 for jasmonic acid. The recoveries ranged from 80 to 105%, with RSDs of 2.8 to 11.2%. The method has been satisfactorily applied for determination of 28 metabolites from Cicer arietinum (L.) and Solanum lycopersicum (L.). It could be an alternative and competitive method of choice that can cheaply and easily perform routine analysis for food quality and targeted metabolomics of chickpea and tomato in response to stressors.     

超高效合相色谱法快速测定保健食品中10种脂溶性维生素

脂溶性维生素作为保健食品重要的功效指标,现有的标准方法存在测定组分少、样品前处理过程复杂、对人员操作能力要求较高等问题,因此建立一种快速、简便、准确,且能同时检测多种常见脂溶性维生素的方法具有重要的现实意义。该研究采用超高效合相色谱(UPC²)建立了同时测定保健食品中维生素A棕榈酸酯、维生素A醋酸酯、维生素E醋酸酯、维生素K₁、α-生育酚、β-生育酚、γ-生育酚、δ-生育酚、维生素D₂、维生素D₃等10种常用脂溶性维生素的方法。样品经含75%二甲基亚砜(DMSO)的水溶液在45 ℃水浴超声15 min破乳,再加入正己烷振摇提取90 min, 3000 r/min离心10 min,取上清液过滤后进行分析。使用LC-Simulator软件对色谱条件进行模拟及优化,选用ACQUITY UPC² Viridis HSS C18 SB色谱柱进行分离,CO₂和乙腈-甲醇(85∶15, v/v)为流动相,梯度洗脱,流速1.9 mL/min,柱温30 ℃,选取10种脂溶性维生素各自的最大吸收波长检测,外标法进行定量。结果表明,10种脂溶性维生素在各自范围内线性关系良好,相关系数(r)均大于0.9997,检出限(LOD)和定量限(LOQ)分别为片剂:0.2~30 μg/g和0.8~75 μg/g,胶囊:0.4~60 μg/g和2~150 μg/g,样品平均加标回收率在96.5%~113.9%之间,RSD均小于4%,精密度、稳定性、重复性测定结果的RSD值也均小于2%。经比较,该方法测定结果与现有的国家食品安全标准基本一致,但该方法简单、快速、灵敏、准确,可满足保健食品中10种脂溶性维生素的检测要求,为保健食品中脂溶性维生素的快速同时检测奠定基础。     

微乳液相色谱法同时测定4种脂溶性维生素

建立了一种新的微乳体系,并成功地应用于微乳液相色谱法(MELC)快速分析脂溶性维生素VA、VD2、VD3和VE。通过对影响分离选择性的主要因素进行考察,得到最佳微乳体系组成为98%(v/v)(50 g/L十二烷基硫酸钠(SDS)-10%(质量分数)正丁醇-1.0%(质量分数)正辛烷-84%水(质量分数))-2%(v/v)乙腈。该微乳体系中,表面活性剂类型和浓度、油相正辛烷的含量、有机添加剂乙腈对脂溶性维生素的分离起到了重要的作用。以Venusil ASB C18色谱柱(150 mm×4.6 mm, 5 μm)为分离柱,流速为0.7 mL/min,检测波长为265 nm,柱温为40 ℃, VA、VD2、VD3和VE在20 min内达到基线分离。4种脂溶性维生素的保留时间和峰面积的相对标准偏差(RSD) (n=5)分别小于2.3%和3.0%; VA、VD2、VD3和VE的线性范围分别为22.0～88.0 mg/L、20.2～81.0 mg/L、24.3～97.2 mg/L和125.0～500.0 mg/L,相应的线性相关系数r2分别为0.9996、0.9994、0.9998、0.9998;检出限(S/N=3)分别为0.37、0.34、0.41和2.12 mg/L。本方法已成功应用于多维元素片(21)中VA与VE的测定,结果令人满意。     

高效液相色谱法同时测定扁桃仁中的水溶性维生素C,B1,B2和B6

建立了用高效液相色谱同时测定扁桃仁中4种水溶性维生素C,B1,B2和B6的方法。以酸水解法处理样品,在Inertsil ODS-3柱(25 cm×4.6 mm i.d., 5.0 μm)上,以0.05 mol/L KH2PO4 (pH 6.0)-甲醇(体积比为70∶30)为流动相进行等度洗脱,检测波长265 nm条件下,对陕西蒲城扁桃仁中的水溶性维生素C,B1,B2和B6的含量进行了同时测定。4种水溶性维生素在其质量浓度为5.0~50 mg/L时线性良好(r=0.9990~0.9997);添加水平为5.0~20.0 mg/kg时,平均回收率为91.77%～99.30%,相对标准偏差(n=3)为0.31%～1.98%。测得陕西蒲城产扁桃仁中维生素B2的含量为4.27～4.53 mg/kg,维生素B1的含量为0.799～0.838 mg/kg,未检出维生素C和维生素B6。该法简便、快速,准确性和重现性均较好,对果仁中水溶性维生素的测定具有一定的参考价值。     

Simultaneous micellar liquid chromatographic analysis of seven water-soluble vitamins: optimization using super-modified simplex

A super-modified simplex (SMS) method has been used to optimize the mobile phase used for separation of seven water-soluble vitamins in multivitamin tablets by gradient micellar liquid chromatography (MLC) with ultraviolet (UV) detection at 254, 295, and 361 nm. Effect of column temperature and addition of organic modifier to the mobile phase on separation efficiency were investigated: the appropriate conditions used were a temperature of 35 degrees C and 1-butanol modifier. The sodium dodecyl sulfate (SDS) concentration, pH, and 1-butanol% in the mobile phase were chosen for simultaneous optimization using the SMS method. The optimum mobile phase was found to be 16 mmol L(-1) (mM) SDS, 0.02 M phosphate buffer, pH 3.6, and a gradient of 3.5-10% (v/v) butanol. The total analysis time for vitamins was 75 min. The analytical parameters including linearity ( r>0.9970), limit of detection (0.12-50 micro g mL(-1)), precision of method (relative standard deviation (RSD) <8.90%), and accuracy obtained by the recovery assay (88-103%) support the usefulness of the proposed method for the determination of the water-soluble vitamins.     

Simultaneous determination of four water-soluble vitamins in fortified infant foods by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

A novel ultra-performance liquid chromatography electrospray ionization tandem triple quadrupole mass spectrometry method for the simultaneous determination of four water-soluble vitamins, including vitamin B5 (VB5), vitamin B8 (VB8), vitamin B9 (VB9), and vitamin B12 (VB12) in fortified infant foods is developed and validated. A reverse phase UPLC separation system consisting of a Waters ACQUITY UPLC BEH C-18 column (2.1 mm x 100 mm i.d., 1.7 microm) and a binary gradient acetonitrile-water mobile phase is applied for the separation of the four water-soluble vitamins. Formic acid is spiked into the mobile phase to enhance the ionization efficiency. Tandem MS-MS analysis is performed in multi-reaction monitoring mode (MRM). Product-ion traces at m/z 220.1 --> 89.9 for VB5, 245.1 --> 227.1 for VB8, 442.3 --> 295.2 for VB9, and 678.9 --> 147.0 for VB12 are used for quantitation of the corresponding vitamins, and traces at m/z 455.5 --> 308.0 are used for methotrexate (internal standard). Limits of quantitation (LOQs) are 0.016, 0.090, 0.020, and 0.019 microg/L for VB5, VB8, VB9, and VB12, respectively. Intra- and inter-day precisions for the determination of the four vitamins are better than 6.84% and 12.26% in relative standard deviations, and recoveries for the four vitamins are in the range of 86.0~101.5%. The developed approach is applied for the determination of the trace amounts of the vitamins in fortified milk powers and fortified rice powers.     

Simultaneous determination of five fat-soluble vitamins in feed by high-performance liquid chromatography following solid-phase extraction

A high-performance liquid chromatography method is developed for the simultaneous determination of menadione, retinyl acetate, cholecalciferol, alpha-tocopherol, and alpha-tocopherol acetate in feed. The present study uses an enzyme to destroy the coating film, ethanol to extract free vitamins, and Oasis HLB cartridges to purify. Vitamins are separated using an Atlantis dC18 column. The mobile phase is methanol-water (98:2 v/v). Detection is performed with a UV-vis detector at 230 and 265 nm. The linearity, accuracy, and repeatability of this method are all satisfactory. Application of the method is suitable for the determination of the fat-soluble vitamins in general feed.     

Simultaneous separation of water‐ and fat‐soluble vitamins in isocratic pressure‐assisted capillary electrochromatography using a methacrylate‐based monolithic column

A method of simultaneous separation of water‐ and fat‐soluble vitamins using pressure‐assisted CEC with a methacrylate‐based capillary monolithic column was developed. In the proposed method, water‐soluble vitamins were mainly separated electrophoretically, while fat soluble‐ones were separated chromatographically by the interaction with a methacrylate‐based monolith. A mixture of six water‐soluble and four fat‐soluble vitamins was separated simultaneously within 20 min with an isocratic elution using 1 M formic acid (pH 1.9)/acetonitrile (30:70, v/v) containing 10 mM ammonium formate as a mobile phase. When the method was applied to a commercial multivitamin tablet and a spiked one, the vitamins were successfully analyzed, and no influence of the matrix contained in the tablet was observed.     

Simultaneous determination and pharmacokinetic study of water-soluble and lipid-soluble components of danshen in rat plasma using HPLC-UV method

A sensitive and specific HPLC-UV method was developed for the simultaneous determination of major active components of danshen in rat plasma. Both water-soluble and lipid-soluble compounds were included, i.e. danshensu, salvianolic acid B and tanshinone IIA. Protocatechuic aldehyde and diazepam were used as internal standards. The chromatographic separation was achieved on a reversed-phase C(18) column by gradient elution using acetonitrile and 0.025% (v/v) phosphoric acid solution as mobile phase, at a flow rate of 1.0 mL/min. Salvianolic acid B, danshensu and internal standards were detected at 281 nm, while the detection of tanshinone IIA was carried out at 272 nm. All calibration curves showed good linearity (r(2) > 0.999) within test ranges. The limit of detection and the limit of quantification for danshensu, salvianolic acid B and tanshinone IIA in plasma were 0.065, 0.043, 0.022, 0.131, 0.085 and 0.044 microg/mL, respectively. This is the first report on the determination and pharmacokinetic study of danshensu, salvianolic acid B and tanshinone IIA in rat plasma and the results indicated that this method was reliable for the determination of the major active components of danshen in rat plasma.     

Simultaneous determination of water-and fat-soluble vitamins by high-performance thin-layer chromatography using an aqueous micellar mobile phase

It is shown that fat-soluble vitamins A and E and water-soluble vitamins B (B₁, B₂, B₆, and B₁₂) can be separated by high-performance thin-layer chromatography using fractional elution. Benzene was used as the first mobile phase, and a 0.02 M aqueous micellar solution of sodium dodecyl sulfate was the second eluant.     

Validated HPLC and HPTLC Method for Simultaneous Quantitation of Amlodipine Besylate and Olmesartan Medoxomil in Bulk Drug and Formulation

Two methods are described for simultaneous determination of amlodipine besylate and olmesartan medoxomil in formulation. The first method was based on the HPTLC separation of two drugs on Merck HPTLC aluminium sheets of silica gel 60 F₂₅₄ using n-butanol: acetic acid: water (5:1:0.1, v/v/v) as the mobile phase. The second method was based on the HPLC separation of the two drugs on the RP-PerfectSil-100 ODS-3–C₁₈ column from MZ-Analysetechnik GmbH, Germany and acetonitrile/0.03 M ammonium acetate buffer (pH = 3) in a ratio of 55:45 as the mobile phase. Both methods have been applied to formulation without interference of excipients of formulation.     

高效液相色谱法同时测定多维元素片中9种水溶性维生素

采用更简便的流动相体系,建立了高效液相色谱法同时测定多维元素片中9种水溶性维生素的快速分析方法。以酸水解与离心的方法处理样品,用C8柱分离,流动相A为0.1%三氟乙酸的水溶液,B为甲醇,梯度洗脱,二极管阵列检测器(DAD)检测。28min内实现了9种水溶性维生素的同时分离测定。各维生素线性关系、精密度、回收率均良好。并使用美国国家标准技术研究院(NIST)的SRM3280多维元素片标准物质对方法进行了确认,运用此法测定了市售多维元素片中的水溶性维生素含量。该法可作为维生素片剂中水溶性维生素分离测定的质控方法。     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

Simultaneous determination of fat- and water-soluble vitamins by microemulsion electrokinetic chromatography

A new procedure was developed for the simultaneous determination of water- and fat-soluble vitamins by microemulsion electrokinetic chromatography. Using microemulsions based on sodium dodecyl sulfate, Brij 35, 1-butanol, and heptane, 10 vitamins were separated (4 water-soluble and 6 fat-soluble) within 35 min. The efficiency of the separation was 800000 theoretical plates (effective capillary length, 40 cm). To enhance the selectivity, 2-propanol was used as a hydrophobizing addition into the microemulsion. The procedure was used for analyzing a kaolin-based vitamin premix. The use of microemulsion as an agent for extracting vitamins from the support considerably simplifies the sample preparation.     

Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection

Water- and fat-soluble vitamins were separated on a MetaChem Polaris C18-A (150 mm×4.6 mm, 3 μm particle size) in a single run using combined isocratic and linear gradient elution with a mobile phase consisting of 0.010% trifluoroacetic acid of pH 3.9 (solvent A) and methanol (solvent B) at the flow rate 0.7 ml min⁻¹. A linear gradient profile (A:B) started at 95:5 and was constant in the first 4 min, then linearly decreased up to 2:98 during the next 6 min, then it was constant in the next 20 min and finally linearly increased up to 95:5 ratio of water phase in the last 5 min of the separation. The most suitable detection wavelength for simultaneous vitamin determination was 280 nm. The method was applied for the solid sample of pharmaceutical preparation (B-Komplex), fortified powdered drinks (multi-vitamin) and food samples. The results were in good agreement with the declared values.     

微乳液相色谱法同时分离7种水溶性维生素

采用微乳液相色谱法同时分离7种水溶性维生素(V_(B1)、V_(B2)、V_(B6)、VB_(12)、叶酸、烟酰胺和VC)。考察了微乳流动相体系中表面活性剂、油相、助表面活性剂的种类以及流动相的pH值、柱温等对水溶性维生素分离的影响。优化后微乳体系的组成为:十二烷基硫酸钠(SDS)/聚氧乙烯月桂醇醚(Brij35)/正丁醇/乙酸乙酯/水(质量比为2∶60∶66∶8∶864)。色谱柱为Agilent TC C18(250 mm×4.6 mm,5μm),柱温为30℃,检测波长为254 nm,流速为0.5mL/min。7种水溶性维生素在20 min内达到基线分离。在4~36 mg/L范围内,7种水溶性维生素的质量浓度与峰面积的相关系数均大于0.999 1。不同添加水平下,V_(B1)、V_(B2)、V_(B6)、VC和烟酰胺的平均回收率为93.9%~102.9%。该方法可用于食品和药品中的多种水溶性维生素的分离、鉴别及快速测定。