Determination of biotin by high-performance liquid chromatography in infant formula, medical nutritional products, and vitamin premixes

A solid-phase extraction sample preparation procedure was developed for use with a high-performance liquid chromatography (HPLC) method for biotin analysis. The HPLC method used a reversed-phase C18 column; chromatography run time was 8.5 min. After eluting from the column, biotin went through postcolumn reaction to form a conjugate with streptavidin-fluorescein isothiocyanate, which was then detected by a fluorescence detector. This method was tested with infant formula, medical nutritional products, and vitamin premix samples.     

Evaluation and single laboratory validation of an on-line turbulent flow extraction tandem mass spectrometry method for melamine in infant formula

This report presents the single-laboratory validation of a method for the determination of melamine in dairy-based products using on-line turbulent flow extraction-tandem mass spectrometry. Liquid or powder test portions were dissolved in water, enriched with (13)C(3)(15)N(3)-Melamine internal standard, followed by protein precipitation and withdrawal of an aliquot for analysis. The turbulent flow method was validated by analyses of liquid and powdered proficiency test portions containing up to 10mg/kg melamine. Accuracy of results ranged from 96 to 106% of the assigned values for the 6 proficiency test portions tested with relative standard deviations of 4-8%. Apparent recoveries based on addition of amino-(15)N(3)-Melamine to prepared test portions were between 98 and 114%. Based on the repeat analysis of a known blank sample the limit of detection and limit of quantification were determined to be 27 and 87 μg/kg, respectively. Additionally, this report demonstrates that turbulent flow chromatography is significantly faster than traditional LC-MS, with sample analysis times of less than 2 min.     

Determination of biotin and folate in infant formula and milk by optical biosensor-based immunoassay

Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using monoclonal antibodies raised against analyte-conjugate. Sample extraction conditions were optimized and antibodies were evaluated for cross-reactivity. Performance parameters included a quantitation range of 2-70 ng/mL, recoveries of 86-102%, agreement against assigned reference values for National Institute of Standards and Technology Standard Reference Material 1846, between-laboratory reproducibility relative standard deviation of 9.1% for biotin and 8.1% for folate, respectively, and equivalence against reference microbiological assay methods for both analytes.     

Comparative evaluation of extraction procedures and method validation for determination of carbohydrates in cereals and dairy products by capillary electrophoresis

This work describes the development and application of two optimized electrolytes: 15 mmol/L sorbate, 0.2 mmol/L CTAB, and 35 mmol/L sodium hydroxide for the determination of fructose, glucose, maltose, maltotriose, and sucrose in cereals and 15 mmol/L sorbate, 0.3 mmol/L CTAB, and 55 mmol/L sodium hydroxide for the determination of fructose, glucose, galactose, lactose, and sucrose in dairy products. Both methods were validated with respect to linearity, limits of detection (using both signal-to-noise ratio and the Eurachem approach), recovery tests, and intra- and inter-day precision exhibiting adequate performance. Additionally, statistically similar results were obtained in a comparative study of extraction procedures for carbohydrates using the AOAC protocol, ultrasound extraction, magnetic stirring, and sample dissolution.     

A rapid, acetonitrile-free, HPLC method for determination of melamine in infant formula

A simple, precise, accurate and validated, acetonitrile-free, reverse phase high performance liquid chromatography (HPLC) method is developed for the determination of melamine in dry and liquid infant formula. The separation is performed on a Kromasil C18 column (150 mm × 3.2 mm I.D., 5 μm particle size) at room temperature. The mobile phase (0.1% TFA/methanol 90:10) is pumped at a flow rate of 0.3 mL min⁻¹ with detection at 240 nm. Melamine elutes at 3.7 min. A linear response (r > 0.999) is observed for samples ranging from 1.0 to 80 μg mL⁻¹. The method provides recoveries of 97.2-101.2% in the concentration range of 5-40 μg mL⁻¹, intra- and inter-day variation in <1.0% R.S.D. The limit of detection (LOD) and limit of quantification (LOQ) values are 0.1 μg mL⁻¹ and 0.2 μg mL⁻¹, respectively.     

Development and validation of an HPLC method for the simultaneous determination of tocopherols, tocotrienols and carotenoids in cereals after solid-phase extraction

The increasing interest in antioxidant properties of cereal and cereal-based products has prompted the development of a simple and reliable HPLC method for the simultaneous determination of important phytochemicals like tocopherols (T), tocotrienols (T3) and carotenoids. Separation was carried out on a Nucleosil 100 C(18) column, 5 μm (250 mm × 4.6 mm) thermostated at 25 °C, using a linear gradient elution system starting with methanol and ending with a mixture of methanol-isopropanol-acetonitrile. All separated compounds including the internal standard (α-tocopherol acetate) were eluted within 16 min and detected by dual detection: fluorescence for tocopherols and tocotrienols at 290 nm excitation and 320 nm emission and UV-vis photodiode array detection for lutein and β-carotene at 450 nm. Detection limits ranged from 0.2 μg/g (β-carotene) to 1.60 μg/g (α-tocopherol). The intra- and inter-assay coefficients of variation were calculated by using cereals with different levels of lipophilic antioxidants. The extraction method involved sample saponification and clean-up by solid-phase extraction (SPE). The extraction recoveries obtained using OASIS HLB SPE cartridges and dichloromethane as eluent were in the range of 90.2-110.1%, with RSD lower than 10%. The method was successfully applied to cereals: durum wheat, bread wheat, rice, barley, oat, rye, corn and triticale.     

Determination of total iodine in infant formula and nutritional products by inductively coupled plasma/mass spectrometry: single-laboratory validation

A single-laboratory validation by inductively coupled plasma-mass spectrometry was developed for the determination total iodine (m/z 127) in infant formula and adult nutritional products. All samples were digested in nitric acid using a closed vessel microwave oven system; Te (m/z 130) was used as an internal standard. To prevent loss of iodine, ammonium hydroxide solution was added to the samples immediately after digestion. The method quantitation limit for total iodine was 0.3 ng/mL, but a practical LOQ was used at 1.0 ng/mL, a concentration at which there was a negligible bias due to nonlinearity. The total iodine concentrations (112-1900 ng/g) in 14 out of 15 nutritional products were within specification limits. Within-day and day-to-day (6 independent days) precision values were < 10% RSD. The observed precision for the overall mean (18 independent days) of a control sample was approximately 4% RSD. In two National Institute of Standards and Technology standard reference materials, total iodine results were within certified limits. Sample spike recoveries for all 15 nutritional products were 92-105%. The data show that a conventional microwave oven digestion procedure can be used to prepare samples for iodine determination. Therefore, this technique is very compatible with other methods being proposed as modern official methods for the analysis of minerals in nutritional products.     

Development,validation and application of an HPLC method for phenol electrooxidation products in the presence of chloride

A high-performance liquid chromatography (HPLC) method was developed and validated to determine phenol and potential intermediates from hydroxylation (hydroquinone, benzoquinone, catechol) and hypochlorination (2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 2,3,6-trichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol) pathways during electrooxidation in the presence of chloride. A Hypersil ODS column (150 mm L × 4.6 mm I.D.) was used for the separation. The best separation was achieved when using a time variant gradient between a water mobile phase (with 0.1% formic acid adjusted to pH 3.0 with 0.1 mM sulfuric acid) and an organic phase (90:5:5 by volume mixture of acetonitrile:methanol:acetic acid). The flow rate was 0.8 mL min^?1 and UV absorbance was monitored at 270, 280, 290 and 300 nm, choosing the wavelength of strongest response for each compound. The intra- and inter-day accuracy and precision were tested using five replicates each day on three consecutive days.     

Determination of niacin in infant formula by solid-phase extraction/liquid chromatography: peer-verified method performance-interlaboratory validation

This paper reports the results of the interlaboratory peer validation study of AOAC Peer-Verified Method (PVM) 1:2,000 for the determination of niacin in infant formula by solid-phase extraction/liquid chromatography. We have used a Data Quality Objectives (DQO) approach to address not only method variability and robustness but also accuracy of data through the use of an appropriate reference material in conjunction with the interlaboratory validation study. Our DQO included the following: (1) statistical agreement of analytical results and quantitative recovery between 2 collaborating laboratories; (2) the repeatability relative standard deviation (RSDr) values and the HORRAT (Horwitz ratio) obtained (1.07), which satisfied the criteria of the Horwitz "limits of acceptability" at the analyte level present; (3) validation of lack of interference; and (4) accuracy agreement within assigned values for a certified reference material. National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1846 Infant Formula, with a certified value of 63.3 +/- 7.6 microg/g for niacin content, was used as a test material for collaborative study and accuracy assessment. Niacin values obtained by the originating laboratory were 59.7 +/- 4.0 microg/g (95% confidence interval [CI] = 1.4 microg/g with a relative standard deviation [RSD] of 6.7%) and by the peer laboratory were 56.6 +/- 6.6 microg/g (95% CI = 4.1 microg/g, with an RSD of 11.7%). Statistical evaluation using the means equivalence test showed that nicotinic acid values obtained by the peer laboratory were equivalent to those values obtained by the originating laboratory. Linear calibration curves and quantitative recovery were obtained. Integration of the PVM process with a readily available certified reference material gives the user confidence in the accuracy of the data generated by the method through traceability to the reference material used.     

Rapid method for determination of protein content in cereals and oilseeds: validation, measurement uncertainty and comparison with the Kjeldahl method

The objective of this research was to test suitability of the Dumas combustion method to completely substitute the Kjeldahl method in routine laboratory determination of crude protein content in cereals and oilseeds. The validation of the method demonstrated that it is able to determine crude protein content in cereals and oilseeds in an efficient and accurate manner, with a detection limit w(N) = 0.006%, quantification limit w(N) = 0.019%, repeatability precision RSD _r = 0.41%, intra-laboratory reproducibility precision RSD _R = 0.74%, trueness, expressed in terms of bias b = 0.43%, and linear response between (2.36–19.2) mg N. Measurement uncertainty, expressed as relative expanded uncertainty (coverage factor k = 2, confidence level 95%), was calculated from validation data (U _rel = 2.24%). In order to examine the relationship between two methods, 15 cereal grain and oilseed samples were analyzed using Dumas and Kjeldahl procedure. The Kjeldahl procedure gave slightly lower w(N) values than the Dumas procedure: w _K(N) = 0.9905 w _D(N) = 0.0376 (R ²  = 0.9996). Relative standard deviations and results of homogeneity test obtained during analysis of complex cereal products (cereal breakfast and muesli bars) show that the Dumas combustion method may be less suitable for analysis of such samples compared to Kjeldahl method.     

Simultaneous determination of free carnitine and total choline by liquid chromatography/mass spectrometry in infant formula and health-care products: single-laboratory validation

A single-laboratory validation study was conducted for a liquid chromatographic/mass spectrometric (LCIMS) method for the simultaneous determination of the free carnitine and total choline in milk-based infant formula and health-care products. The sample preparation used for both carnitine and choline was adapted from AOAC Official Method 999.14, with an acidic and enzymatic hydrolysis of esterified forms of choline. Carnitine and choline were quantified by ion-pair chromatography with single-quadrupole MS detection, using their respective deuterated internal standards. The repeatability relative standard deviation was < or =2.5 and 2.1%, respectively, for carnitine and choline. The intermediate reproducibility relative standard deviation was <4.7 and 2.4%, respectively, for carnitine and choline. The ranges of the average product-specific recoveries were 92-98 and 94-103%, respectively, for carnitine and choline. Choline concentration determined in infant formula reference material SRM 1846 was in agreement with the reference value. The proposed method was compared with the enzymatic methods for a range of products; good correlation (r = 0.99) was obtained, although a significant bias was observed for both analytes. The method, with a short chromatographic run time (7 min), is convenient for routine analysis to enhance analytical throughput and is a good alternative to enzymatic assays.     

Single-laboratory validation of a liquid chromatographic/tandem mass spectrometric method for the determination of free and total carnitine in infant formula and raw ingredients

A reversed-phase liquid chromatographic/tandem mass spectrometric method was developed and validated for the determination of free carnitine (FC) and total carnitine (TC) in infant formula and raw ingredients. Preparation of samples for FC determination required only nonvolumetric dilution in water, followed by filtration. Determination of TC in whey-based samples required a common saponification procedure, which hydrolyzed the acylcarnitines to FC. L-Carnitine standards were prepared in water at 3 concentrations (7.5, 30, and 60 ng/mL). L-Carnitine-d3 was incorporated in the sample and standard preparations as a stable isotope internal standard (ISTD), which helped to normalize signal suppression due to fouling of the ionization source, or matrix effects. A short C8 column (50 x 2.1 mm id) was used for liquid chromatography, with mobile phases consisting of 0.1% heptafluorobutyric acid in water and methanol. L-Carnitine and the ISTD eluted at about 2 min, and the injection-to-injection cycle time was about 11 min. Multiple-reaction monitoring was used for quantitation by monitoring the ion transitions m/z 162 > 103 and 162 > 85 for L-carnitine, and m/z 165 > 103 and 165 > 85 for the ISTD. The values for overall method precision for a whey-based infant formula, a protein hydrolyzate infant formula, and a water-soluble vitamin/amino acid premix were relative standard deviations of 1.32, 1.86, and 3.33%, respectively. The overall mean recoveries were between 97.3 and 102.8%. Additionally, the method results showed good correlation with those obtained for a radioenzymatic assay done in-house.     

Development and independent laboratory validation of a simple method for the determination of paraquat and diquat in potato, cereals and pulses

A new sensitive, fast and robust method for the determination of paraquat and diquat residues in potatoes, cereals and pulses is presented. Different extraction conditions (solvent, time and temperature) have been evaluated using barley grain, potatoes and dry lentils containing incurred residues of diquat and paraquat. The finalised procedure involves extraction with a mixture of methanol/water/hydrochloric acid at 80?°C and analysis by liquid chromatography–tandem mass spectrometry. Diquat D4 and Paraquat D6 internal standards were added to the test portions prior to extraction. A small-scale inter-laboratory validation of the developed method for diquat and paraquat using potato and barley samples was conducted by three laboratories. The precision and accuracy of the method were determined from recovery experiments (five replicates) at 0.01 and 0.1?mg?kg^?1. The recoveries obtained (n?=?180) were in the range of 92–120?% with associated relative standard deviation (RSD) between 1.4–10?% for all compound/commodity/spiking concentration combinations.     

HILIC-MS/MS method for the quantitation of nucleotides in infant formula and adult nutritional formula: First Action 2011.21

Official Method 2011.21 is for the quantitation of the following nucleotides: adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), and inosine 5'-monophosphate (IMP) in infant formula and adult/pediatric nutritional formula. It uses hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Preparation of the internal standards was conducted using centrifugal ultrafiltration and the standards are AMP- (13)C10, (15)N5; GMP-(13)C10, (15)N5; UMP-(13)C9, (15)N2; and15 CMP- (13)C9, (15)N3. Data were collected by using multiple reaction monitoring of the product ions of protonated molecules of the five nucleotides generated by positive-electrospray ionization. The HILIC conditions were conducted with ammonium formate (30 mmol/L) in water (pH 2.5, adjusted with formic acid) and methanol. The LOD and LOQ of the standard solution were 0.005-0.01 and 0.01-0.03 microg/mL, respectively. Recovery data were collected for intraday and interday testing and ranged from 98.1 to 108.9% with an RSD of 0.7-5.4%. The analytical range of the method is between 0.04 to 5 microg/mL for standard solution.     

Optimization and validation of an enantioselective method for a chiral drug with eight stereo-isomers in capillary electrophoresis

Highly selective capillary electrophoresis (CE) screening methods were applied to find a satisfactory separation of a chiral drug with eight stereoisomeric compounds. The initial separation conditions were further optimized using response surface modelling by applying a Box-Behnken experimental design. This approach resulted in a rapid and efficient optimization of the buffer concentration, the concentration of two cyclodextrins, and the run voltage, in order to obtain final separation conditions of the method. Further optimization and validation of the system in terms of sensitivity and robustness resulted in a method that is suitable for quality control release purposes.     

Development and validation of an ion‐exchange chromatography method for heparin and its impurities in heparin products

An anion‐exchange liquid chromatography method for the determination of heparin and its impurities (dermatan sulfate and oversulfated chondroitin sulfate) was developed using chemometric‐assisted optimization, including multivariate experimental design and response surface methodology. The separation of heparin, dermatan sulfate, and oversulfated chondroitin sulfate (R_s above 2.0) was achieved on a Dionex RF IC IonPac AS22 column with a gradient elution of 10–70% of 2.5 M sodium chloride and 20 mM Tris phosphate buffer (pH 2.1) at a flow rate of 0.6 mL/min and UV detection at 215 nm. Method validation shows good linearity (r > 0.99), acceptable precision (%relative standard deviations <11.4%) and trueness (%recovery of 92.3–103.9%) for all analytes. The limits of detection for dermatan sulfate and oversulfated chondroitin sulfate are equivalent to 0.11% w/w (10.5 μg/mL) and 0.07% w/w (7.2 μg/mL), while the limits of quantification are 0.32% w/w (31.5 μg/mL) and 0.22% w/w (22.0 μg/mL) relative to heparin, respectively. The method is specific for heparin, dermatan sulfate, and oversulfated chondroitin sulfate without interference from mobile phase and sample matrices and could be used for accurate quantitation the drug and its impurities in a single run. Applications of the method reveal contents of heparin between 90.3 and 97.8%. Dermatan sulfate and oversulfated chondroitin sulfate were not detected in any of the real‐life samples.