Determination of vitamin K1 in emulsified nutritional supplements by solid-phase extraction and high-performance liquid chromatography with postcolumn reduction on a platinum catalyst and fluorescence detection

Determination of small amounts of vitamin K1 (0.8 microg/g) in nutritional supplements with high fat content (20 mg/g) was performed by solid-phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection after reduction on a platinum oxide catalyst. The concentration ratio of plant oils to vitamin K1 (0.8 microg/g) was about 25,000:1. A sample solution was applied to a solid-phase extraction cartridge and vitamin K1 was eluted with ethanol, followed by HPLC. The proposed method was simple, rapid (analysis time: ca. 12 min), sensitive [detection limit: ca. 0.1 pg per injection (100 microl) at a signal-to-noise ratio of 3:1], highly selective and reproducible [relative standard deviation: ca. 1.3%. (n=5)]. The calibration graph of vitamin K1 was linear in the range of 0-2 pg per injection (100 microl). Recovery of vitamin K1 was over 90% by the standard addition method.     

Determination of streptomycin residue in cucumber and Chinese cabbage by high-performance liquid chromatography with postcolumn derivatization and fluorometric detection

A sensitive and accurate method was developed for the determination of streptomycin using HPLC followed by postcolumn derivatization and fluorometric detection. The analyte was extracted, using aqueous solution from cucumber and Chinese cabbage, by a two-step SPE procedure. The extraction, cleanup, and chromatography conditions were optimized, and the performance of the analysis method was evaluated. The conditions of chromatography were as follows: the separation was performed on a C18 column; the isocratic mobile phase consisted of acetonitrile and a mixed solution containing 10 mM sodium 1,2-naphthoquinone-4-sulfonate and 0.4 mM sodium 1-heptanesulfonate (25+75, v/v); and the flow rate was 1 mL/min. The fluorescence detector was set at an excitation wavelength of 263 nm and an emission wavelength of 435 nm. The calibration curve was linear over the range of 50-2000 ng/mL, with a correlation coefficient of 0.9995. The LOD and LOQ were 10 and 30 ng/g, respectively, in both cucumber and Chinese cabbage. The method was validated for selectivity, linearity, precision, and accuracy. The intraday and interday precision and accuracy were within 10%. The mean recoveries from spiked samples were more than 75%, with RSD lower than 10%.     

Determination of free and reversibly-bound sulfite in selected foods by high-performance liquid chromatography with fluorometric detection

A rapid and accurate method for measuring low part-per-million levels of free and reversibly-bound sulfites in selected foods by using high-performance liquid chromatography (HPLC) with fluorometric detection was developed. Sulfites were extracted with sodium tetrachloromercurate solution and determined by HPLC-fluorescence spectrometry. During the HPLC analysis, the sample extract was reacted with sodium hydroxide to liberate the reversibly-bound sulfite and subsequently separated from other interferences by a size exclusion column. The effluent was then reacted with o-phthalaldehydelammonium chloride reagent to form a fluorescing 1-sulfonatoisoindole derivative and analyzed by a fluorescence detector. The method has been applied to a variety of foods and food products, with no significant interference encountered in matrixes such as soy products, cabbage, broccoli, brassica, ginger, fungus, mushroom, mandarin peel, potato chips, and biscuits. It was shown to have a broad linear range of 0.01 to 0.4 mg/L as SO2. The spike recoveries of sodium sulfite, sodium metabisulfite, and formaldehyde-sodium bisulfite adduct at the 5 to 10 mg/kg level in the tested food matrix were within 80-120%, and the limit of detection was 5 mg/kg. Laboratory of Government Chemist Reference Material LGC7111 (potato powder) was used to assess the accuracy of the method. The expanded measurement uncertainty of the method at 95% confidence level was estimated to be 17%.     

Determination of vitamin K1 isomers in foods by liquid chromatography with C30 bonded-phase column

Vitamin K1 was determined in a variety of foods by using reversed-phase liquid chromatography with a C30 column followed by post-column reduction to the fluorescent hydroquinone derivatives. Lipids were removed by lipase digestion, followed by single extraction into hydrocarbon, and the protocol was extended to selected natural and processed foods. Biologically active trans- and inactive ci-vitamin K1 isomers were measured individually to evaluate the true nutritional status of the products. Method performance parameters confirmed the validity of the technique. The use of the triacontyl-bonded C30 phase for selective phylloquinone isomer measurement extends previously validated AOAC Method 999.15 for vitamin K1 in milk and infant formula to a wider range of foods important in the human diet. The cis-vitamin K1 isomer contributes up to about 15% of total phylloquinone in certain foods.     

Determination of vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection

A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.     

Determination of adriamycin and daunorubicin in urine by high-performance liquid chromatography with laser fluorometric detection

A separation and detection scheme is presented for the determination of the antitumor drugs adriamycin and daunorubicin in human urine. Separation is accompished by reversed-phase high-performance liquid chromatography and the drugs are detected down to the low picograms level by laser excited fluoresence using a unique fiber optic based flow-cell. Excellent detector selectivity and linearity are reported, and some of the factors influencing the performance of the detector are discussed. Possible extension of the procedure to other biologically important determinations are mentioned.     

柱后还原-高效液相色谱法同时测定调制乳粉中维生素K1和维生素K2

建立了一种同时检测调制乳粉中维生素K₁和维生素K₂的柱后还原-高效液相色谱-荧光检测方法。样品用水溶解，经脂肪酶酶解，2.5 mol/L氢氧化钠溶液和乙醇溶液皂化，正己烷萃取，氮吹浓缩后，用甲醇复溶。通过Xbridge C₁₈色谱柱分离，锌粉还原柱柱后还原，荧光检测器检测，激发波长为326 nm，发射波长为432 nm，外标法定量。结果表明，维生素K₁在0.0025~2.0 μg/mL、维生素K₂在0.01~2.0 μg/mL范围内线性关系良好，相关系数均大于0.999，维生素K₁和维生素K₂的检出限分别为0.07 μg/100 g和0.24 μg/100 g，定量限分别为0.2 μg/100 g和0.8 μg/100 g；方法的加标回收率为80.39%~94.39%，精密度为0.85%~3.98%。该方法灵敏度高，重复性好，结果准确，适用于调制乳粉中维生素K₁和维生素K₂的分析检测。     

Determination of phylloquinone (vitamin K1) by high performance liquid chromatography with UV detection and with particle beam-mass spectrometry

A reversed-phase PB LC-MS method was used for the determination and unequivocal identification of vitamin K(1) in some vegetable samples. The method was also applied to the identification of other constituents in the food analyzed. LC-MS experiments were carried out in both EI and negative-ion CI modes. Preliminary sensitivity studies on vitamin K(1) showed a detection limit of 2 ng (signal-to-noise ratio=3) in negative-ion operation and in single-ion monitoring at m/z 450. Quantitative results obtained with LC-MS for the extracts of four vegetables were compared with those of LC-UV analyses performed at 247 nm.     

Simultaneous determination of vitamin K1, vitamin K1 2,3-epoxide and menaquinone-4 in human plasma by high-performance liquid chromatography with fluorimetric detection

A highly sensitive method for measuring endogenous vitamin K1, menaquinone-4 (which is one of the K2 vitamins) and vitamin K1 2,3-epoxide in human plasma was developed, based on high-performance liquid chromatography with coulometric reduction and fluorimetric detection, following extraction from plasma and purification on a Sep-Pak silica cartridge. The detection limits of vitamin K1, menaquinone-4 and vitamin K1 2,3-epoxide were 5, 5 and 8 pg per injection for the standard substances and 30, 30 and 50 pg/ml in human plasma, respectively.     

Determination of oxytetracycline, tetracycline, and chlortetracycline in milk by liquid chromatography with postcolumn derivatization and fluorescence detection

A multiresidue method for isolation and liquid chromatographic determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) in milk is presented. The sensitivity of the method is adequate to meet the needs of regulatory agencies. The European Community established 100 micrograms/kg as the maximum residue limit (MRL) in milk for TC, CTC, and OTC. Recoveries exceeded 80% for all tetracyclines at all levels, with good precision. Correlation coefficients of standards curves for individual tetracyclines isolated from fortified samples ranged from 0.991 for CTC to 0.998 for OTC. Other antibiotics that might interfere with analysis did not interfere with elution times of OTC, TC, and CTC. The procedure is rapid, precise, and quantitative and requires minimal preparation and minimal use of organic solvents. It can be applied to routine surveillance programs. We can prepare 10 samples for analysis in about 1.45 h.     

Determination of streptomycin residues in food by solid-phase extraction and liquid chromatography with post-column derivatization and fluorometric detection

A reliable and sensitive procedure is presented for the analysis of streptomycin (STP) in food of animal origin, like meat, milk and honey. The method is based on a separation by ion-pair liquid chromatography with β-naphthoquinone-4-sulfonate (NQS) postderivatization and fluorescence detection. The clean-up of the extract is done by solid-phase extraction, firstly with a cation-exchange cartridge and secondly with an octadecyl cartridge. The selectivity is very good and not many interfering peaks are observed for various food matrices. The streptomycin recovery of the total procedure is superior to 80%. The procedure is quantitatively characterized and repeatability, linearity, detection and quantification limits are very satisfactory. A special focus is given to STP residues in honeys and a survey on 64 commercial honeys is presented. For honey analysis, the HPLC method is compared with an immunoassay test (ELISA), and the possibility of using this test for screening with and without solid-phase extraction clean-up is also discussed.     

Determination of carbaryl as its primary metabolite, 1-naphthol, by reversed-phase high-performance liquid chromatography with fluorometric detection

Carbaryl and its hydrolysis product, 1-naphthol, are determined simultaneously, and in a mixture of other pesticides, by reversed-phase high-performance liquid chromatography (HPLC). Fluorometric detection affords a higher degree of selectivity than absorbance detection, while providing detection limits of 1.0 ng ml(-1) and 1.4 mg ml(-1) for 1-naphthol and carbaryl, respectively. The described method requires no enrichment of samples and includes a simple hydrolysis step. Additionally, enhancement of fluorescence detection signal intensity is explored through HPLC solvent composition effects as well as through formation of cyclodextrin inclusion complexes. The method described in this report is a selective and fast alternative to the currently accepted U.S. Environmental Protection Agency (EPA) method of detection carbaryl in ground water.     

Determination of vitamin A in pharmaceutical preparations by high-performance liquid chromatography with diode-array detection

Summary A very simple high-performance liquid chromatographic method for determination of Vitamin A in pharmaceutical preparations without the need for saponification was developed. A reversed-phase (Nova-Pack C₁₈, 4 m) column was used with a mobile phase of acetonitrile-tetrahydrofuran-water (55378) and a flowrate of 1.5 ml/min. Sample treatment only consisted of the extraction of retinol acetate content from capsules or tablets with methanol. Total extraction was achieved by shaking vigorously with the aid of magnetic stirring for three hours at room temperature. No change of solvent is necessary to introduce the sample in the chromatographic system. This method is suitable for routine quantification of Vitamin A.     

Determination of bovine blood oleandrin by high-performance liquid chromatography and postcolumn derivatization

A high-performance liquid chromatographic procedure with a postcolumn fluorescence derivatization is developed for the analysis of oleandrin in bovine blood. Oleandrin is separated by an octadecylsilane-bonded column with a mobile phase containing dehydroascorbic acid. The effluent of the column is mixed with concentrated hydrochloric acid and passed through poly(tetrafluoroethylene) tubing maintained at 70 degrees C. The resultant fluorophores are detected at 465 nm with excitation at 348 nm. Simple solid-phase extraction using Sep-Pak tC2 is effective for sample purification. We found the minimal detectable quantity of oleandrin in plasma to be 1.5 ng/mL at a signal-to-noise ratio of 3:1.