Development of a green chromatographic method for determination of colorants in food samples

A green chromatographic analytical method for determination of Tartrazine, Brilliant Blue and Sunset Yellow in food samples is proposed. The method is based on the modification of a C18 column with a 0.25% (v/v) Triton X-100 aqueous solution at pH 7 and in the usage of the same surfactant solution as mobile phase without the presence of any organic solvent modifier. After the separation process on the chromatographic column, the colorants are detected at 430, 630 and 480 nm, respectively. The chromatographic procedure yielded precise results and is able to run one sample in only 8 min, consuming 15.0 mg of Triton X-100 and 38.8 mg of phosphate. When the flow rate of the mobile phase is 1 ml min⁻¹ the retention times are 2.1, 3.6 and 7.0 min for Tartrazine, Brilliant Blue and Sunset Yellow, respectively; and all peak resolutions are ca. 2. The analytical curves present the following linear equations: area = 7.44 10⁵ + 2.71 10⁵ [Tartrazine] (R = 0.998, n = 7); area = 1.09 10⁵ + 3.75 10⁵ [Brilliant] (R = 0.9995, n = 7) and area = −7.34 10⁴ + 2.33 10⁵ [Sunset] (R = 0.998), n = 7) and, the limits of detection for Tartrazine, Brilliant Blue and Sunset Yellow were estimated as 0.125, 0.080 and 0.143 mg l⁻¹. When the proposed method is applied to food samples analysis, precise results are obtained (R.S.D. < 5%, n = 3) and in agreement with those obtained by using the classical spectrophotometric method. The traditional usage of organic solvent as mobile phase in HPLC is not used here, which permits to classify the present method as green.     

Simultaneous spectrophotometric determination of fat-soluble vitamins in multivitamin pharmaceutical preparations

A spectrophotometric method is proposed for the simultaneous determination of vitamins A, D and E in multivitamin pharmaceutical preparations. This is based on multiple linear regression. Most vitamins are directly extracted from the preparations into n-hexane. Microen-capsulated vitamin A preparations require pretreatment of de-encapsulation before the vitamin is extracted. The wavelength range to be used for each preparation and the optimum spectral mode (absorbance or first-derivative) has been chosen in order to assure correct quantitation and avoid interferences from other absorbing species also extracted by n-hexane. The results obtained were validated by simultaneous HPLC analyses for accuracy and precision.     

Simultaneous microemulsion liquid chromatographic analysis of fat-soluble vitamins in pharmaceutical formulations: Optimization using genetic algorithm

An environmentally benign and simple method has been proposed for separation and determination of fat-soluble vitamins using isocratic microemulsion liquid chromatography. Optimization of parameters affecting the separation selectivity and efficiency including surfactant concentration, percent of cosurfactant (1-butanol), and percent of organic oily solvent (diethyl ether), temperature and pH were performed simultaneously using genetic algorithm method. A new software package, MLR-GA, was developed for this purpose. The results indicated that 73.6 mM sodium dodecyl sulfate, 13.64% (v/v) 1-butanol, 0.48% (v/v) diethyl ether, column temperature of 32.5 °C and 0.02 M phosphate buffer of pH 6.99 are the best conditions for separation of fat-soluble vitamins. At the optimized conditions, the calibration plots for the vitamins were obtained and detection limits (1.06–3.69 μg mL⁻¹), accuracy (recoveries > 94.3), precision (RSD < 3.96) and linearity (0.01–10 mg mL⁻¹) were estimated. Finally, the amount of vitamins in multivitamin syrup and a sample of fish oil capsule were determined. The results showed a good agreement with those reported by manufactures.     

Selection of a ternary eluant for the determination of fat-soluble vitamins in multivitamin pharmaceutical preparations by high-performance liquid chromatography

A procedure for selecting a ternary eluant for the determination of the synthetic fat-soluble vitamins E acetate, A acetate, K₃, and D₂ in Hexavit, Undevit, and Hendevit multivitamin pharmaceutical preparations was proposed, and the composition of then-hexane-l,2-dichloroethane-n-butanol (86.42 :13.42: 0.16, vol %) eluant was optimized.     

A gas chromatographic flow method for preconcentration and determination of vitamins D2 and D3 in pharmaceutical preparations

Summary A continuous-flow method that combines on-line preconcentration and isolation with gas chromatography for the direct determination of vitamins D₂ and D₃ in oily solutions is reported. A silica gel column permits preconcentration and isolation of analytes from the vitamin D matrix, although some triglyceride (ca. 25%) is also retained. To overcome problems associated with the low volatility of triglycerides, their retained fraction is further transesterified with potassium methylate to fatty acid methyl esters after elution. The proposed method was applied to the determination of vitamin D in pharmaceutical preparations.     

Development and validation of a thin-layer chromatographic method for determination of chloramphenicol residues on pharmaceutical equipment surfaces

A thin-layer chromatographic (TLC) method with densitometric quantitation using the absorption reflectance mode at 280 nm was developed and validated for the determination of chloramphenicol residues in controlling pharmaceutical equipment cleanliness. Simulated samples at residue levels 0.5, 1, and 1.2 mg/m2 were prepared by spreading the calculated amount of chloramphenicol solution on a 10 dm2 stainless steel surface. After evaporation of the solvent, the residue was removed by 2 methanol-wetted cotton swabs, which were then extracted with methanol. The extract was applied on a high-performance TLC (HPTLC) silica gel F254 plate together with standards ranging from 10 to 60 ng. Plates were developed in a horizontal developing chamber from both sides (36 applications per plate) by using n-hexane-ethyl acetate (35 + 65, v/v) as developing solvent. The mean recovery (n=6) at 1 mg/m2 was 95.8%, and the coefficient of variation was 5.8%. The absolute detection limit was 3 ng, and the quantitation limit 10 ng. The method detection limit was 0.3 mg/m2 by swabbing 2.5 dm2 and 0.075 mg/m2 by swabbing 10 dm2. Chloramphenicol was stable on the plate 2 h before and 24 h after development. Additionally, it was stable during 7 days storage on the cotton swabs in the solvent at room temperature and in diluted standard solution stored in darkness at 4 degrees C. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from the stainless steel surface areas of 2.5 to 10 dm2, and an acceptable residue limit of 1 mg/m2.     

Development and validation of a reversed-phase column liquid chromatographic method for the determination of five cephalosporins in pharmaceutical preparations

A new, simple, rapid, and precise RP-HPLC method has been developed and validated for the determination of five cephalosporins, namely, cefalexin, cefoperazone, ceftriaxone, ceftazidime, and cefepime. The method has been applied successfully for simultaneous determination of cefalexin in a binary mixture with sodium benzoate in a suspension, and cefoperazone in a binary mixture with sulbactam in vials. Chromatographic separation was achieved on a Waters microBondapak C18 column (250 x 4.6 mm id, 10 pm particle size) using the mobile phase monobasic potassium phosphate (50 mM, pH 4.6)-acetonitrile (80 + 20, v/v) with UV detection. A flow rate of 1 mL/min was applied. Linearity, accuracy, and precision were found to be acceptable over the concentration range of 30-300, 3-30, and 15-120 microg/mL for the studied cephalosporins, sodium benzoate, and sulbactam, respectively. The optimized method proved to be specific, robust, and accurate for QC of the cited drugs in their pharmaceutical preparations.     

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.     

Development and validation of a high-performance thin-layer chromatographic method for determination of ofloxacin residues on pharmaceutical equipment surfaces

An HPTLC method with densitometric quantification using fluorescence at 313 nm was developed and validated for the determination of ofloxacin residue in controlling pharmaceutical equipment cleanliness. Simulated samples at a residue level of 1 mg/m2 were prepared by spreading the calculated amount of ofloxacin solution on 1, 5, and 10 dm2 stainless steel surfaces. After evaporation of the solvent, the residue was removed by two ethanol wetted cotton swabs, which were thereafter extracted with the mixture of ethanol and Na2EDTA-water solution at pH 11 for 15 min with sonication. The extract and standards were applied on HPTLC silica gel 60 plates and then developed in a horizontal developing chamber from both sides using ethanol-conc. ammonia (4+1, v/v) as the mobile phase. The mean recovery (n=6) at 1 mg/m2 from 1, 5, and 10 dm2 was 95.3, 88.6, and 89.7% with the CV values 3.78, 4.41, and 4.97%, respectively. The absolute detection limit was 0.6 ng and the quantitation limit was 2 ng, but it was shown that these can be improved by immersion of the developed plate into a solution of liquid paraffin-n-hexane (1+2, v/v) to approximately 0.25 and 0.9 ng, respectively. The LOD of the method using detection without paraffin-n-hexane was 3, 0.6, and 0.3 microg/m2 by swabbing 1, 5, and 10 dm2, respectively. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from stainless steel surface areas of 1 to 10 dm2 with acceptable residue limit/surface of 1 mg/m2.     

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C₁₈ analytical column (250 mm × 4.6 mm i.d., 5 μm particle size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min⁻¹. The ultraviolet (UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95 min, respectively. The calibration graph was ranged from 2.50 to 20.00 μg mL⁻¹, while detection and quantitation limits were found to be 0.48 and 1.61 μg mL⁻¹, respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, %E_r, was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.     

Validation of a liquid chromatographic method for determination of tacrolimus in pharmaceutical dosage forms

An accurate, simple, and reproducible liquid chromatographic method was developed and validated for the determination of tacrolimus in capsules. The analysis is performed at room temperature on a reversed-phase C18 column with UV detection at 210 nm. The mobile phase is methanol-water (90 + 10) at a constant flow rate of 0.8 mL/min. The method was validated in terms of linearity, precision, accuracy, and specificity by forced decomposition of tacrolimus, using acid, base, water, hydrogen peroxide, heat, and light. The response was linear in the range of 0.09-0.24 mg/mL (r2 = 0.9997). The relative standard deviation values for intra- and interday precision studies were 1.28 and 2.91%, respectively. Recoveries ranged from 98.06 to 102.52%.     

Optimization of solvent selectivity for the chromatographic separation of fat-soluble vitamins using a mixture-design statistical technique

Summary A systematic approach, using a mixture-design statistical technique, has been developed for selecting the optimum mobile phase for the separation of fat-soluble vitamins in reversed-phase high-performance liquid chromatography. A quaternary mixture of methanol, acetonitrile, tetrahydrofuran and water was used as mobile phase. Retention time and peak width were recorded in ten runs augmented with five replicates and the data were subsequently fitted to special cubic polynomial models. The resulting mathematical equations enabled prediction of resolution over the entire parameter space. Contour plots of minimum effective resolution and maximum retention time as a function of mobile phase composition are presented and discussed. Visual inspection of these plots provides an overview of the quality of the separation and the analysis time required for each possible mobile-phase composition with n the parameter space. It is demonstrated that the methodology followed was an important tool which enabled the taking of informed decisions necessary for selection of the optimum mobile phase for a chromatographic separation. A combination ofR _S minimum andt _R maximum as optimization criteria in a multicriteria decision-making plot using pareto-optimality concept is discussed. This combination enabled visual demonstration of the compromise between separation quality and the economics of analysis time. Our methodology has been compared with the common used technique of ‘overlapping resolution mapping’.     

High-performance liquid chromatographic separation of the fat-soluble vitamins in cod liver oil and feeds

A MicroPak-CN column with a methylene chloride—chloroforms-n-hexane (3:2:15) mobile phase separates the fat-soluble vitamins in cod liver oil and formulated feed. Powdered feed samples are dissolved in n-hexane—chloroform—ethanol (6:3.5:0.5) and centrifuged before injection. The oil is dissolved in the extraction solvent or mobile phase before injection. The naturally occurring vitamins are identified from their absorption spectra by stopped-flow spanning of the individual vitamins.     

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.     

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。     

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%.     

Development of validated stability-indicating chromatographic method for the determination of fexofenadine hydrochloride and its related impurities in pharmaceutical tablets

ABSTRACT: A simple reversed phase high performance liquid chromatographic method with diode array detector (HPLC-DAD) has been developed and subsequently validated for the determination of fexofenadine hydrochloride (FEX) and its related compounds; keto fexofenadine (Impurity A), meta isomer of fexofenadine (Impurity B), methyl ester of fexofenadine (Impurity C) in addition to the methyl ester of ketofexofenadine (Impurity D). The separation was based on the use of a Hypersil BDS C-18 analytical column (250 × 4.6 mm, i.d., 5 μm). The mobile phase consisted of a mixture of phosphate buffer containing 0.1 gm% of 1-octane sulphonic acid sodium salt monohydrate and 1% (v/v) of triethylamine, pH 2.7 and methanol (60:40, v/v). The separation was carried out at ambient temperature with a flow rate of 1.5 ml/min. Quantitation was achieved with UV detection at 215 nm using lisinopril as internal standard, with linear calibration curves at concentration ranges 0.1-50 μg/ml for FEX and its related compounds. The optimized conditions were used to develop a stability-indicating HPLC-DAD method for the quantitative determination of FEX and its related compounds in tablet dosage forms. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. Complete separation was achieved for the parent compounds and all degradation products. The method was validated according to ICH guidelines in terms of accuracy, precision, robustness, limits of detection and quantitation and other aspects of analytical validation.     

High-performance liquid chromatographic method for routine determination of vitamins A and E and beta-carotene in plasma.

A simple and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the routine determination of vitamins A and E and beta-carotene in plasma (or serum) with wavelength-programmed ultraviolet-visible absorbance detection is described. A 200-microliters aliquot of serum or plasma sample, after deproteinization with ethanol, and containing tocopherol acetate as internal standard, was extracted with butanol-ethyl acetate. Sodium sulphate was added for dehydration. Analytes of extracted samples were found to be stable for at least four days. A 10-microliters aliquot of this organic extract was used for HPLC analysis. The mobile phase was methanol-butanol-water (89.5:5:5.5, v/v) and the flow-rate was set at 1.5 ml/min. The analytes of interest were well separated from other plasma constituents within 22 min at 45 degrees C. The lowest detection limits of vitamins A and E and beta-carotene were 0.02, 0.5 and 0.1 microgram/ml, respectively. The recovery and reproducibility of the present method were around 90%. The method is sensitive, specific and can be used for epidemiological studies and for routine determination of vitamin deficiency. Several important factors that may affect the analysis are also discussed in this paper.