Simultaneous determination of 20 pharmacologically active substances in cow's milk, goat's milk, and human breast milk by gas chromatography-mass spectrometry

This paper reports a systematic approach to the development of a method that combines continuous solid-phase extraction and gas chromatography-mass spectrometry for the simultaneous determination of 20 pharmacologically active substances including antibacterials (chloramphenicol, florfenicol, pyrimethamine, thiamphenicol), nonsteroideal anti-inflammatories (diclofenac, flunixin, ibuprofen, ketoprofen, naproxen, mefenamic acid, niflumic acid, phenylbutazone), antiseptic (triclosan), antiepileptic (carbamazepine), lipid regulator (clofibric acid), β-blockers (metoprolol, propranolol), and hormones (17α-ethinylestradiol, estrone, 17β-estradiol) in milk samples. The sample preparation procedure involves deproteination of the milk, followed by sample enrichment and cleanup by continuous solid-phase extraction. The proposed method provides a linear response over the range of 0.6-5000 ng/kg and features limits of detection from 0.2 to 1.2 ng/kg depending on the particular analyte. The method was successfully applied to the determination of pharmacologically active substance residues in food samples including whole, raw, half-skim, skim, and powdered milk from different sources (cow, goat, and human breast).     

Liquid chromatographic determination of vitamin D in fortified milk

A method for the determination of vitamins D2 + D3 in fortified milk is described. Vitamins D2 and D3 are extracted from the saponified sample and converted to isotachysterols with antimony trichloride. The isotachysterols are quantitated using liquid chromatography with ultraviolet detection at 301 nm, which is the absorption maximum. At this wavelength other materials present in the sample do not interfere with the analysis of isotachysterols and therefore a cleanup step is avoided. Recoveries of vitamin D added to skim milk were 98.1% (SD 5.3), 96.7% (SD 3.3), and 96.0% (SD 5.1) for samples fortified with 200, 400, and 600 IU/quart, respectively. For whole milk, recoveries were 102.0% (SD 6.5) and 97.1% (SD 3.5) in samples fortified with vitamin D equivalent to 200 and 400 IU/quart, respectively. The detection limit for vitamin D is 40 IU/quart.     

Sample preparation and liquid chromatographic determination of vitamin D in food products

Vitamin D in different fortified foods is determined by using liquid chromatography (LC). Sample preparation is described for fortified skim milk, infant formulas, chocolate drink powder, and diet food. The procedure involves 2 main steps: saponification of the sample followed by extraction, and quantitation by LC analysis. Depending on the sample matrix, additional steps are necessary, i.e., enzymatic digestion for hydrolyzing the starch in the sample and cartridge purification before LC injection. An isocratic system consisting of 0.5% water in methanol (v/v) on two 5 microns ODS Hypersil, 12 X 0.4 cm id columns is used. Recovery of vitamin D added to unfortified skim milk is 98%. The results of vitamin D determination in homogenized skim milk, fortified milk powder, fortified milk powder with soybean, chocolate drink powder, and sports diet food are given.     

Microwave-assisted rapid determination of vitamins a and e in beverages

A new rapid procedure for the determination of vitamins A and E in beverages has been developed and validated. Key steps include a microwave-assisted saponification of the sample and a single-step extraction of the vitamins prior to HPLC analysis. All sample preparation steps are carried out consecutively in the same vial. The vitamins are determined using normal-phase (Si-60) HPLC with fluorescence detection. The method is applicable to beverages with a content of all-trans-retinol >0.14 mg/L and/or a content of alpha-tocopherol >1 mg/L. Recoveries determined by spiking experiments ranged from 91.3 to 106.3%. The precision of the method is characterized by relative standard deviations of <2% for alpha-tocopherol and <5% for all-trans-retinol.     

Inulin determination for food labeling

Inulin and oligofructose exhibit valuable nutritional and functional attributes, so they are used as supplements as soluble fiber or as macronutrient substitutes. As classic analytical methods for dietary fiber measurement are not effective, several specific methods have been proposed. These methods measure total fructans and are based on one or more enzymatic sample treatments and determination of released sugars. To determine inulin for labeling purposes, we developed an easy and rapid anion-exchange high-performance liquid chromatography (HPLC) method following water extraction of inulin. HPLC conditions included an Aminex HPX- 87C column (Bio-Rad), deionized water at 85 degrees C as the mobile phase and a refractive index detector. The tested foods included tailor-made food products containing known amounts of inulin and commercial products (cookies, milk, ice creams, cheese, and cereal bars). The average recovery was 97%, and the coefficient of variation ranged from 1.1 to 5% in the food matrixes. The obtained results showed that this method provides an easier, faster and cheaper alternative than previous techniques for determining inulin with enough accuracy and precision for routine labeling purposes by direct determination of inulin by HPLC with refractive index detection.     

Biochemical fluorometric method for the determination of riboflavin in milk

Methods of analysis of vitamin B2 in foods generally consist of the extraction of the sample, followed by enzymatic hydrolysis and quantitative measurement of the analyte, typically through RP-HPLC. The scope of our work here is to present a soft method to measure the free riboflavin content of a nontransparent and nonhomogeneous matrix such as milk, avoiding any extraction and separation of phases that are required in any published method for determination of the free RBF content in foods. We combine the front-face (FF) measurement of the light emission of milk with the ability of the apo-form of the riboflavin-binding protein (RBP) from chicken egg white to quench the riboflavin fluorescence. Thus, we titrate the RBF present in milk by gradually adding a solution of RBP to the milk sample and measuring, upon each addition, the FF residual emission due to uncomplexed RBF. The RBP binding capability has been measured in the same matrix of the analyte. Our results indicate a concentration of free RBF practically co-incident with the certified value for total B2 vitamin content in reference milk CRM 421. Keywords: Front-face fluorescence; riboflavin; apo-riboflavin-binding protein; milk fluorescence.     

Rapid and sensitive determination of sulfonamide residues in milk and chicken muscle by microfluidic chip electrophoresis

A new, rapid, and sensitive method was proposed for the determination of sulfonamide residues in milk and chicken muscle samples by microchip electrophoresis with laser-induced fluorescence detection. Separation of fluorescamine-labeled sulfonamides was accomplished by using a buffer containing 5 mmol/L boric acid and 1% (w/v) polyvinyl alcohol (PVA). The pH, amount of PVA, and concentration of boric acid in the running buffer were found to have great influence on the separation. By optimizing these conditions, the separation of four sulfonamides, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, and sulfanilamide, was achieved within 1 min with limits of detection (S/N = 3) of 0.2-2.3 μg/L, which are well below the maximum residue limit. The proposed method also exhibited very good repeatability; the relative standard deviations for both within-day and between-day measurements were ≤3.0%. With a simplified sample pretreatment protocol, fast determination of sulfonamides in real samples was successfully performed with standard addition recoveries of 93.3-100.8 and 82.9-92.3%, respectively.     

Disposable cartridge extraction of retinol and alpha-tocopherol from feedstuffs

Automated determination of fat-soluble vitamins by modern methods such as liquid chromatography is hampered by the initial extraction step. A simple technique is proposed that allows an appreciable increase in the actual rates of determination. Feedstuff samples are first hydrolyzed in an aqueous alcohol (mainly methanol)-potassium hydroxide solution. Instead of extracting retinol and alpha-tocopherol from the hydrolysis solution by an organic solvent, an aliquot of the solution is mixed with a small volume of a strong antioxidant solution (ascorbic acid) and pipetted onto a kieselguhr disposable cartridge where it is adsorbed. Retinol and alpha-tocopherol are eluted with isooctane at normal pressure. The proposed method has been compared with conventional techniques on many feed samples.     

Determination of taurine in infant formulas using ultrafiltration and cation-exchange chromatography

A fast and simple method for determination of taurine in infant formulas has been developed. The sample preparation uses disposable ultrafiltration cartridges to remove protein and clarify the sample. Hydrolysis is avoided, simplifying the procedure and increasing efficiency. One mL sample is centrifuged in a cartridge for 45 min. The filtrate is diluted with pH 2.2 citrate buffer and injected into a high performance amino acid analyzer. A cation-exchange column (sodium phase) is used with a single buffer eluant and an isocratic chromatographic program. Colorimetric detection is performed following post-column ninhydrin reaction. Chromatographic resolution from other ninhydrin-positive compounds is excellent. Average recoveries for 3 levels of spike for various products were 100-102%. Precision is 1-3% RSD, depending on product. Linearity, specificity, and ruggedness are excellent. The method is applicable to quality control testing of milk-based, soy-based, and prehydrolyzed protein-based infant formulas in the ready-to-use, concentrate, and powder forms. A variety of commercially available infant formulas from different manufacturers were analyzed and all were found to contain taurine levels comparable to human milk. Some human milk and cow's milk samples were also analyzed and results compare well with literature values.     

A quantitative stable-isotope LC-MS method for the determination of folic acid in fortified foods

A stable-isotope liquid chromatography-mass spectrometry (LC-MS) assay was developed for the quantitative determination of folic acid in fortified foods. Folic acid was extracted from food samples into a phosphate buffer, purified on a C-18 Sep-Pak cartridge, and analyzed by LC-MS in the negative ion mode using electrospray ionization. The analyte was quantified using (13)C(5)-folic acid as an internal standard. The coefficient of variation for the precision of the method was 5.6% based on the analysis of four sample replicates. The accuracy of the method was assessed using a standard method of addition of folic acid to a shredded whole-wheat cereal. The quantitative determination of folic acid in this matrix was linear over 1 order of magnitude having a concentration range of 2.4 to 24 microg/g of food (or 0.05 to 0.5 microg of analyte injected into the LC-MS). The overall quantitative efficiency of the method was evaluated using a standard reference material (infant formula SRM 1846). The method was applied to the determination of folic acid in several test samples (fortified breakfast cereals), and the values were in accord with the manufacturer's claim. This method advances a LC-MS technique for the determination of folic acid in fortified foods based on stable-isotope dilution methodology. The specificity of the technique and quantitative accuracy of the method in various food substrates suggests that the method may be adapted for routine analysis in other fortified foods.     

Liquid chromatographic analysis of riboflavin vitamers in foods using fluorescence detection

The analysis of free riboflavin (RF) and its two coenzymes, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD), is optimized using reversed phase liquid chromatography with fluorescence detection. The stationary phase was amide-based and endcapped with trimethylsilyl, and the isocratic mobile phase consisted of a 10:90 v/v acetonitrile/phosphate buffer (pH 5). Peaks were identified by the retention characteristics and fluorescence spectra. Detection limits were 0.03, 0.05, and 0.24 ng for RF, FMN, and FAD, respectively. The vitamins were extracted using acetonitrile and the phosphate buffer. The procedure was applied to the determination of B2 vitamers in different types of food such as milk and soy-based infant formulas, beer, fruit juices, and honey of different types. Most B2 vitamin appeared as RF, while the coenzymes were present in lower amounts. The method was validated using two certified reference materials, and results within the certified range were obtained.     

Detection and identification of beta-lactam residues in milk using a hybrid biosensor

A novel application of a hybrid biosensor is here employed as an analytical method for the detection and presumptive identification of beta-lactam residues in milk. The method is based on measurements of carbon dioxide (CO2), the production of which is related to the microbial growth of the test microorganism Bacillus stearothermophilus var. calidolactis. The presence of beta-lactams in milk inhibits microbial growth and, consequently, the CO2 production rate. The analysis is based on the variation of CO2 between a milk sample spiked with beta-lactams and a twin milk sample containing beta-lactams plus a broad spectrum beta-lactamase, using an electrochemical device of biosensor. A blank milk sample is included as control. The result is obtained starting from the first 120 min. Moreover, the ability to recognize all of the beta-lactams speeds the total time of analysis when chemical identification and quantification are required. The analytical method appears to be adequate for milk control for qualitative screening purposes, complying with the requirements stated in Decision 2002/657/EC.     

利用电学阻抗技术检测巴氏杀菌乳的物理特性

阻抗特性作为宏观的电学参数可以实现对牛乳的理化性质评估。在20 Hz～12 MHz频率区间采用带螺旋测微仪的平行板测量系统连接阻抗分析仪研究了3类巴氏杀菌的全脂乳、低脂乳和脱脂乳在不同稀释比例及开封后室温储藏时的阻抗特性。提出巴氏杀菌乳的RC等效电路并采用ZSimpWin软件拟合得到了乳样的电学元件参数,发现低脂乳的稀释比例和其等效电阻呈指数关系,决定系数为0.8337(P<0.05)。各类巴氏杀菌乳的阻抗幅值随频率的提高而降低,相位角则呈现先减少后增加的趋势,其阻抗特性与脂肪含量无明显规律性,但与稀释比例存在显著性关系,20 Hz和1033 Hz时的全脂乳和脱脂乳的相位角与稀释比例具有良好的指数回归关系,决定系数为0.9887和0.9493(P<0.01),而低脂乳于11.4 kHz下相位角与稀释比例呈线性回归,决定系数为0.9846(P<0.01)。室温下开封储藏的乳品内部发生复杂的生化反应且伴随有机物分解,全脂乳和脱脂乳存放时间与其阻抗特性无显著性关系,但低脂乳在207 Hz的激发电场频率下的相位角与储藏时间呈现对数回归关系,决定系数为0.889(P<0.05)。该研究为实现生产中巴氏杀菌乳的浓度和储藏时间分析评估提供了快速而可靠的方法。     

Rapid direct determination of lead in evaporated milk by anodic stripping voltammetry without sample pretreatment

The method presented describes the direct determination of lead in evaporated milk in which the milk ashing step prior to analysis is eliminated. Digital instrument readout units are microgram Pb/mL milk. Total analysis time after instrument calibration is less than 3 min per sample. Range of the method is 0.05-1.0 ppm lead in milk, and precision of the method expressed by relative standard deviation of duplicate pairs ranged from 30% at 0.1 micrograms/mL to 3% at 1.0 micrograms/mL of lead in milk. The method compares favorably with the AOAC official first action anodic stripping voltammetric method (25.074). In addition, the method appears to work equally well for skim evaporated milk, sweetened condensed milk, and nonfat powdered dry milk when the latter two are reconstituted with water according to product label instructions. Recovery and interference studies are presented.     

Rapid determination of total cholesterol in homogenized milk

A rapid method that is amenable to automation has been developed for the determination of total cholesterol in homogenized milk. The milk sample is saponified in ethanolic KOH in the presence of an internal standard, cholestane. Cholesterol and the internal standard are then isolated by solid-phase extraction on a nonpolar adsorbent and eluted with organic solvent. The evaporated extract is derivatized and analyzed by capillary gas chromatography. Average recovery of cholesterol acetate added to milk prior to saponification was 95%. The average relative standard deviation for repeated analyses was 2%. The limit of detection for this method is 2 mg/100 g. Twenty samples can be analyzed by one analyst in a normal work day if the gas chromatograph is equipped with an autosampler. This method has been compared with a modified AOAC method for the determination of total cholesterol. At a confidence level of 95%, no difference was observed between the 2 methods.     

Rapid Methods for Milled Rice Surface Total Lipid and Free Fatty Acid Determination

Total lipids and free fatty acid (FFA) determination is widely used in the food industry to assess the quality of milled rice. An improved rapid ambient temperature isopropanol (IPA) extraction method to determine milled rice surface lipid was more effective than Soxhlet solvent extraction. The improved method was probably due to better extraction of polar lipids and antioxidants by IPA. A colorimetric method to determine FFA requiring only 30 μL of sample is also described. The new technique provides results similar to those obtained using the slower, conventional acid‐base titration method. The colorimetric FFA method requires a smaller sample size, has greater precision, and is more objective. The new methods are particularly suitable for industrial use in providing rapid results for large numbers of samples.     

Photoprotection of vitamins in skimmed milk by an aqueous soluble lycopene-gum Arabic microcapsule

Riboflavin (Rf)-mediated photosensitized degradation of vitamins A and D3 in skimmed milk under illumination with a white fluorescence lamp was studied by using the HPLC technique. The photosensitized degradation of both vitamins followed first-order kinetics, and the temperature effect on the observed photodegradation rate constant allowed the determination of the activation energy Ea as being 4 and 16 kcal/mol for vitamins A and D3, respectively. The addition of lycopene microencapsulated by spray-drying with a gum arabic-sucrose (8:2) mixture (MIC) produced a reduction of ca. 45% in the photosensitized degradation rate of both vitamins. Front-face fluorescence experiments showed the same photoprotection factor in the degradation of Rf itself, indicating that the photodegradation mechanism involved Rf-mediated reactive species, such as the excited triplet state of Rf, 3Rf*, and/or singlet molecular oxygen, 1O2. The interaction of both 3Rf* and 1O2 with MIC was evaluated in aqueous solutions by using laser-induced time-resolved absorption or emission spectroscopy, and the contribution of an inner-filter effect in the presence of MIC in skimmed milk was evaluated by diffuse reflectance spectroscopy. The main operating mechanism of photoprotection is due to the deactivation of 3Rf* by the proteic component of gum arabic; thus, gum arabic based microcapsules could be used to improve the photostability of milk during its storage and/or processing under light.     

Gas-liquid chromatographic determination of captan and two metabolites in milk and meat

A gas-liquid chromatographic (GLC) method has been developed for the determination of captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) and 2 metabolites, tetra-hydrophthalimide (THPI) and tetrahydrophthalamic acid (THPMA), in milk and meat. The sample is extracted with ethyl acetate and is cleaned up by acetonitrile partition and silica gel chromatography where captan, THPI, and THPMA are separated. Captan is directly determined by GLC. THPI and THPMA are separately derivatized in an acetone solution of pentafluorobenzyl bromide. The resultant derivatives are purified separately on an Al2O3 column and quantitated by GLC, using an electron capture detector. Recoveries from milk samples fortified at 0.02-10 ppm ranged from 71 to 102%; recoveries from meat samples fortified at 0.04-10 ppm ranged from 75 to 99%.     

A quantitative method for the determination of cyclopropenoid fatty acids in cottonseed, cottonseed meal, and cottonseed oil (Gossypium hirsutum) by high-performance liquid chromatography

Cyclopropenoid fatty acids (CPFAs), found in cottonseed, have been shown to have detrimental health effects to susceptible livestock. Previous quantitative analytical methods for the determination of CPFAs expressed these acids in terms of their relative abundance with respect to other fatty acids in the oil, necessitating the concurrent analysis of other fatty acids. The proposed analytical method describes the quantitation of three relevant CPFAs for cotton (malvalic acid, sterculic acid, and dihydrosterculic acid) in cottonseed in micrograms per gram fresh weight of sample. The method involves extraction of the oil, saponification, and derivatization of the free fatty acids with 2-bromoacetophenone to give the phenacyl esters. These esters are then separated by dual-column reverse-phase high-performance liquid chromatography and quantitated via external standards. This is the first method to include external calibration standards for CPFAs and, as such, is capable of direct quantification with no further data conversion required. CPFA data generated from the analysis of cottonseed, cottonseed meal, and cottonseed oil produced in the United States in 2002 are presented.     

Automatic method for the determination of Folin-Ciocalteu reducing capacity in food products

In the present work, an automatic flow procedure based on multi-syringe flow injection analysis was developed for the assessment of Folin-Ciocalteu reagent (FCR) reducing capacity in several types of food products using gallic acid as the standard. Different strategies for mixing of sample and reagent were tested (continuous flow of FCR, merging zones, and intercalated zones approaches); lower reagent consumption and higher determination throughput were attained for the merging zones approach (100 microL of sample+100 microL of FCR). The application of the proposed method to compounds with known antioxidant activity (both phenolic and nonphenolic) and to samples (wines, beers, teas, soft drinks, and fruit juices) provided results similar to those obtained by the conventional batch method. The detection limit was 0.6 mg L-1, and the determination frequency was about 12 h-1. Good repeatability was attained (RSD<1.3%, n=10).