高效液相色谱法测定蜂产品中链霉素残留量

采用双试剂柱后衍生法,在C18柱上以0.01mol L1 庚烷磺酸钠 乙腈(65 35)为流动相,以荧光检测器(λex=263nm,λem=447nm)检测蜂蜜中链霉素的残留量,优化了蜂蜜样品的前处理方法和高效液相色谱条件,包括流动相组成和流速、衍生化试剂的处理、样品前处理过程中淋洗液和提取液的用量等,并选用三氯乙酸溶液(1 1)作为沉淀剂,建立了一种较好的链霉素检测方法。     

High-performance liquid chromatographic determination of clenbuterol and cimaterol using post-column derivatization.

A general high-performance liquid chromatographic method for the simultaneous and rapid determination of cimaterol and clenbuterol is described. Solid samples, such as animal tissues, faeces and feeding-stuffs, are extracted with dilute acid saturated with ethyl acetate. The resulting extracts or liquid samples, such as urine, plasma, blood and bile, are purified via Chem Elut columns. Separation is achieved by ion-pair chromatography on a Nova-Pak C18 column, and highly specific detection is obtained with an adapted version of the post-column derivatization described previously for the determination of clenbuterol in urine and animal tissues. Detection limits for liquids and solids are 0.1 ng/ml and 0.2 ng/g, respectively. The results are in complete agreement with analysis by high-performance thin-layer chromatography and gas chromatography-mass spectrometry, applied for confirmation after the same sample pretreatment. With this simple method, complete analysis of a liquid sample needs about 30 min and, even without an automatic sampler, 40 samples can be completely analysed in one day. This method has been used on a routine scale for nearly two years.     

Analysis of barbiturates by micro-high-performance liquid chromatography with post-column photochemical derivatization

This study attempts to combine the advantages of microcolumn high-performance liquid chromatography (μ-HPLC) with those of post-column photochemical derivatization in barbiturate analysis. Some barbiturates are photochemically unstable, leading to photoproducts that show maximum absorption at 270 nm and not the typical one at ≈220 nm. For this purpose, a laboratory-built photoreactor has been developed to work with μ-HPLC instruments. Its performance is satisfactory in the forensic analysis of barbiturates.     

Ion-exchange liquid chromatographic analysis of bisphosphonates by on-line post-column photochemical reaction and spectrophotometric detection

A simple ion-exchange high-performance liquid chromatographic method was developed and employed for the analysis of bisphosphonate compounds in dosage formulations using on-line post-column photochemical reactions. The method used molybdate as the post-column reagent to react with the photolyzed bisphosphonate to form phosphomolybdate for enhanced spectrophotometric detection. A bisphosphonate compound, 2-thioethane-1,1-bisphophonic acid, was selected to evaluate the separation using both isocratic and gradient elution methods, along with the effects of other experimental parameters including mobile phase composition, flow-rate and post-column reagent concentration. The gradient elution method showed improved resolution and detection sensitivity compared to the isocratic elution method. The optimized gradient method was simple, reproducible, and specific to bisphosphonate compounds. It was successfully employed for the stability study of the bisphosphonate compound in pharmaceutical dosage formulations.     

Liquid chromatographic analysis of vitamin B6 in reconstituted infant formula: collaborative study

A liquid chromatographic (LC) method was validated for the determination of total vitamin B6 in infant formula. Total vitamin B6 was quantified by converting the phosphorylated and free vitamers into pyridoxine. Pyridoxine was determined by ion pair reversed-phase LC with fluorescence detection. The method was subjected to an AOAC collaborative study involving a factory-manufactured, milk- and soy-based infant formula. Each was spiked at 3 concentrations in the range of 0-1 microg/g and sent as blind duplicate to participant laboratories. Nine laboratories returned valid data which were statistically analyzed for outliers and precision parameters. The repeatability relative standard deviation (RSD(r)) ranges were 2.0-4.0 and 3.5-5.9% for fortified milk- and soy-based formulas, respectively. The reproducibility relative standard deviation (RSD(R)) ranges were 8.2-8.4 and 6.7-11.2% for fortified milk- and soy-based formulas, respectively. HORRAT values ranged from 0.42 to 0.53, indicating that the precision of the method is acceptable. The mean RSD(r):RSD(R) values were 0.60 and 0.55 for milk- and soy-based formulas, respectively. As expected, RSDs for the unfortified samples were higher, but their HORRAT values (0.81 and 2.06) helped define a realistic limit of quantitation as 0.05 microg/g. Recovery data were quantitative and varied between 81.4 and 98.0% (mean = 89.8%) for each of 6 spiked materials.     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxin B1 in cattle feed: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B1 in cattle feed at a possible future European regulatory limit (1 ng/g). The test portion was extracted with acetone-water (85 + 15), filtered, diluted with water, and applied to an immunoaffinity column. The column was washed with water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase liquid chromatography (RP-LC) and detected by fluorescence after post column derivatization (PCD) involving bromination. PCD was achieved with either pyridinium hydrobromide perbromide (PBPB), used by 14 laboratories, or an electrochemical cell and addition of bromide to the mobile phase, used by 7 laboratories. Both derivatization techniques were not significantly different when compared by the t-test; the method was statistically evaluated for all laboratories together (bromination and PBPB). The cattle feed samples, both spiked and naturally contaminated with aflatoxin B1, were sent to 21 laboratories in 14 different countries (United States, Japan, and Europe). Test portions were spiked at levels of 1.2 and 3.6 ng/g for aflatoxin B1. Recoveries ranged from 74 to 157%. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 5.9 to 8.7%. The relative standard deviation for reproducibility (RSDR) ranged from 17.5 to 19.6%. The method showed acceptable within- and between-laboratory precision for this matrix, as evidenced by HORRAT values, at the target levels of determination for aflatoxin B1. No major differences in RSD were observed, showing that the composition of the feeds was not a factor for the samples tested and that the method was applicable for all materials used.     

Determination of sulfamethazine in swine and cattle feed by reversed-phase liquid chromatography with post-column derivatization: collaborative study

A liquid chromatographic (LC) method for the analysis of sulfamethazine (SMT) in complete swine and cattle feed was collaboratively studied. The method uses post-column derivatization with dimethylaminobenzaldehyde and detection at 450 nm. To 5g finely ground feed, extractant (0.2N HCl + 1.5% diethylamine in 25% methanol), and internal standard solutions are added, and the SMT is extracted by shaking for 1 h. Clarified extract (high-level sample extract diluted to a target concentration of ca 5.5 microg/mL) is chromatographed on a Cla reversed-phase LC column with acetonitrile-2% acetic acid (17 + 83) mobile phase. Sulfamerazine is used as an internal, or surrogate standard to correct for variable recovery of sulfamethazine from a variety of feed matrixes. Six Youden matched-pair samples were sent to 10 collaborators in Korea, Canada, and the United States. Label claims on the commercial feeds ranged from 0.0077 to 0.22% SMT. The SMT mean recovery as determined from the 5 samples with known analyte content was 99.8%. The within-laboratory relative standard deviation (repeatability) ranged from 0.28 to 4.72%. Among-laboratory (including within-laboratory) relative standard deviation (reproducibility) ranged from 1.26 to 4.87%. The authors recommend the method for AOAC INTERNATIONAL Official First Action status.     

High-performance liquid chromatographic method for determining trichothecene mycotoxins by post-column fluorescence derivatization

The method described here is based on a separation of deoxynivalenol, nivalenol and fusarenon-X on a C18 column using aqueous acetonitrile, and successive post-column fluorescence derivatization involving an alkaline decomposition to form formaldehyde and modified Hantzsch reaction with methyl acetoacetate and ammonium acetate (lambda ex = 370 nm and lambda em = 460 nm). By this method, 5-10 ng of the standard trichothecenes could be determined. By employing a clean-up procedure with a florisil column and a Sep-Pak CN cartridge, 61.4-96.9% recoveries were obtained for deoxynivalenol and nivalenol added to corn, wheat and barley at concentration levels of 0.05-1 ppm.     

A sensitive post-column photochemical derivatization/fluorimetric detection system for HPLC determination of bisphosphonates

A new reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method has been developed for the determination of the following bisphosphonic acids: alendronic acid (ALEN), etidronic acid (ETID), ibandronic acid (IBAN) and risedronic acid (RISE). Separation was achieved on a C₁₈ column using a mixture of 50 mmol L⁻¹ borate buffer pH 9.0 containing 0.25 mmol L⁻¹ tetrabutylammonium chloride and 0.5 mmol L⁻¹ EDTA and acetonitrile (97:3) as the mobile phase. The sensitive detection of the above bisphosphonic acids was based on their oxidation to orthophosphate by the on-line peroxydisulfate-assisted photolysis followed by post-column reaction with molybdate to yield phosphomolybdate. This subsequently reacted with thiamine to generate thiochrome and, finally, the fluorescence of thiochrome was measured at 440 nm with excitation at 375 nm. The developed method is precise with a mean relative standard deviation of 1.3%, sensitive (with a detection limit at the nmol L⁻¹ level), accurate, specific, rapid (analysis time approximately 13 min) and inexpensive because to the low cost of the reagents. The assay was applied to the analysis of the four bisphosphonic acids in commercial dosage formulations, in which the excipients did not interfere with the determination. The method was also applied to the determination of etidronate, risedronate and ibandronate in human urine. Sample preparation involves precipitation of the analytes from urine along with endogenous phosphates such as calcium salts by addition of calcium chloride at alkaline pH and dissolution of the precipitate in 0.05 mol L⁻¹ ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid.     

High-performance liquid chromatographic assay of phosphate and organophosphorus pesticides using a post-column photochemical reaction and fluorimetric detection

A sensitive method for the post-column reaction detection of organophosphorus pesticides is described. The method relies on photolysis of the organocompounds by irradiation with a low-pressure mercury lamp (main spectral line, 254 nm) in the presence of peroxydisulfate. The resultant orthophosphate was reacted with molybdate to form molybdophosphoric acid, which subsequently reacted with thiamine to generate thiochrome. Finally, the fluorescence intensity of thiochrome was measured at 440 nm with excitation at 375 nm. Factors affecting the rate of these reactions were optimized so that its contribution to the total band-broadening was negligible.

This detection system was used for the determination of phosphate, acephate and methamidophos, which were separated on an ODS column by isocratic reversed phase chromatography with acetonitrile–water as the mobile phase. A linear relationship between analyte concentration and peak area was obtained within the range 0.016–7.0 μg ml⁻¹ with correlation coefficients greater than 0.9995 and detection limits between 4 and 12 ng ml⁻¹. Intra- and inter-day precision values of about 1.2% R.S.D. (n = 10) and 2.1% R.S.D. (n = 30), respectively, were obtained.

Pesticide residues below ng ml⁻¹ levels could be determined in environmental waters when a preconcentration device was coupled on-line with the HPLC system. Detection limits as low as 0.01 ng ml⁻¹ were achieved for only 250 ml of sample. In the analyses of vegetables and grains, the detection limit was about 1 μg kg⁻¹.     

Fluorimetric determination of cyclofenil using photochemical derivatization

In this work, a fluorimetric approach for the determination of cyclofenil, 4-4′-(cyclohexylidenemethylene)bis(phenylacetate), is presented. The method was based on the intense fluorescence (250/410 nm) observed after a UV photochemical treatment of cyclofenil. The influence of the pH and solvent system and UV exposure time on the fluorescence magnitude was studied. Optimization of parameters was made using experimental design (factorial design and central composite design). Limit of detection (3S_b/m) were estimated to be 1.1 × 10⁻⁸ mol L⁻¹ with the linear dynamic range extending up to 8 × 10⁻⁵ mol L⁻¹. This analytical approach was tested through the analysis of a commercial pharmaceutical formulation. In this case, tests enabled an average recovery of 98.3 ± 3.9% (for n = 9) using the analytical curve. The identification of the fluorescent derivative is proposed based on results achieved from GC-MS.     

Trace analysis of sugars by HPLC and post-column derivatization

Summary A HPLC system with post-column derivatization for the quantitative analysis of sugars in complex matrices is described. As reagent a 0.2% solution of thymol in concentrated sulfuric acid has been used. The reaction is sensitive with reducing as well as non reducing sugars whereas sugar alcohols are discriminated. With this reagent and separation of sugars at high pH values with an anion exchange column it is possible to detect sugars in the ng range. Hence, it is possible to characterize wines not only by their fructose and glucose content but also by differences in the distribution of the other not fermentable sugars like trehalose, arabinose, galacturonic and glucuronic acid.     

Liquid chromatographic method for the determination of calcium cyanamide using pre-column derivatization.

A specific and stability-indicating high-performance liquid chromatographic (HPLC) method has been developed for the analysis of calcium cyanamide in bulk material and dosage form. Calcium cyanamide in samples was converted into dansyl cyanamide. A muBondapak C18 column was employed for HPLC with 0.01 M sodium phosphate (pH 6.3)-acetonitrile (75:25, v/v) as the mobile phase. The proposed HPLC method was validated for linearity, specificity, accuracy and reproducibility.     

High-performance liquid chromatographic determination of proteins by post-column fluorescence derivatization with thiamine reagent.

A post-column derivatization method for the high-performance liquid chromatography of peptides and proteins giving a fluorescence intensity proportional to the number of peptide bonds is described. Peptide bonds were chlorinated with hypochlorite and the N-chlorite formed was allowed to react with thiamine to give fluorescent thiochrome. This method was applied the determination of membrane-forming proteins of microorganisms.     

Liquid chromatographic determination of maleic hydrazide in technical and formulated products: collaborative study

Fourteen collaborating laboratories assayed maleic hydrazide (MH), 6-hydroxypyridazin-3(2H)-one, in technical and formulated products by reversed-phase liquid chromatography (LC) with sulfanilic acid as an internal standard. The active MH in the samples (6 lots) ranged from 16% (expressed as the potassium salt) to 98% (MH in the technical). A small amount of 1 M KOH was added to the technical MH and analytical standards to create the potassium salt of the analyte which is soluble in water. Test samples and standards were extracted with water containing the internal standard before analysis by LC on a C8 column with an ion-pairing eluting solution and UV detection at 254 nm. The concentration of MH was calculated by comparing the peak area response ratios of the analyte and the internal standard with those in the analytical standard solution. Eleven laboratories weighed each test sample twice with single analysis. Three laboratories weighed each sample once and made duplicate injections on the LC system. The data were analyzed using the 11 laboratories' results. A second data analysis was done including all laboratory results using a Youden pair approach, selecting one of 2 duplicate assay values randomly for each laboratory and sample. In the first data analysis, the repeatability standard deviation ranged from 0.07 to 1.39%; reproducibility standard deviation ranged from 0.22 to 1.39%. In the second data analysis (using all laboratory data), repeatability standard deviation ranged from 0.09 to 0.86%; reproducibility standard deviation ranged from 0.22 to 1.31%.     

Liquid chromatographic determination of residual nitrite/nitrate in foods: NMKL collaborative study

Nitrite and nitrate are used as additives in the food industry to provide color and taste and to control undesirable gas and flavor production by anaerobic bacteria by virtue of their antimicrobial properties. The analytical method that has been widely used to determine nitrite and nitrate involves the use of toxic cadmium. In response to a request from the Nordic Committee on Food Analysis, a study was performed to obtain an alternative chromatographic method to determine residual nitrite and nitrate in meat products. The study was done in 3 stages: (1) comparative evaluation of the performance of 3 liquid chromatographic methods, (2) internal validation of the selected ion chromatographic method, and (3) a collaborative study in which 17 laboratories from European countries participated. Furthermore, the applicability of the method to matrixes other than meat and meat products was demonstrated. The results of the collaborative study show that the European Prestandard prENV 12014-4 is well suited for the determination of nitrite and nitrate in different foods (e.g., meat products, vegetables, baby food, and cheese). The limits of detection for nitrite and nitrate ions are 1 and 10 mg/kg, respectively. Recoveries of residual nitrite/nitrate ranged from 96 to 108%. Repeatability and reproducibility were satisfactory.