A collaborative study to validate in vitro assays for protein digestibility assessment using pH-drop and pH-stat methods.

Although free amino acids (FAAs) represent a significant component of ripened cheeses and can provide useful information for their characterization, no inter-laboratory validated analytical method exists which allows a reliable comparison of data obtained by different laboratories and the adoption of quality control schemes based on FAA pattern. The objective of the present work was to test the effectiveness of an analytical protocol for the determination of the FAA composition of cheese and to verify the adequateness of this type of analysis for quality control procedures of Grana Padano PDO cheese as well as for research purposes. After an initial test to compare performances of ion-exchange chromatography (IEC) and HPLC techniques, an inter-laboratory collaborative study (seven laboratories, four samples) was organized to validate an IEC method with post-column ninhydrin derivatization and using l-norleucine as an internal standard. Determined amounts of individual FAA ranged from 8 to over 1380 mg/100 g cheese, with relative standard deviation of repeatability (RSDr) ranging from 0.5 to 4.6%, and relative standard deviation of reproducibility (RSDR) ranging from 1.3 to 9.9% for FAA concentrations over 100 mg/100 g. For lower concentrations, RSDr and RSDR were about thrice as high. On the basis of the results of this investigation, at present, the validated method is adopted as the official method for the determination of FAA patterns in the quality control of Grana Padano PDO cheese.

Determination of Deoxynivalenol in Poultry Feed by Three Gas Chromatographic Detection Techniques

To validate an LC-MS/MS method using a strong anion exchange cartridge for simultaneous determination of fumonisin B1, B2 and B3 in corn, an inter-laboratory study was performed in 9 laboratories using one fumonisin-negative corn sample, three spiked corn samples (FB1: 100-1,000 μg/kg, FB2 and FB3: 10-100 μg/kg) and two naturally contaminated corn samples. The recoveries were in the ranges of 79.7-87.2% for FB1, 78.6-103.2% for FB2 and 80.1-92.8% for FB3. The relative standard deviations for repeatability (RSDr) ranged from 3.7 to 8.0% for FB1, from 2.6 to 15.3% for FB2 and from 4.3 to 9.7% for FB3. The relative standard deviations for reproducibility (RSDR) for FB1, FB2 and FB3 were in the ranges of 6.3-10.1%, 5.9-18.7% and 9.3-16.0%, respectively. The HorRat values for all analytes ranged from 0.2 to 0.9. The difference of the trueness between the two kinds of commercially available anion exchange cartridges used in this study was not significant (p>0.05). Surveillance for fumonisins in corn grits was performed using the validated method. All of the samples were contaminated with fumonisins and the mean concentrations for FB1, FB2 and FB3 were 118.1, 37.3 and 17.9 μg/kg, respectively. These results indicated that the method for simultaneous determination of FB1, FB2 and FB3 in corn was successfully developed and validated.

A gas chromatography-mass spectrometry method for the analysis of phencyclidine (PCP) in urine was subjected to an inter-laboratory study. The collaborative study followed the guidelines provided by the Association of Official Analytical Chemists International. Ten laboratories participated, analyzing 3 samples of PCP-spiked urine as blind duplicate. The repeatability relative standard deviation (RSDr) and the reproducibility relative standard deviation (RSDR) were between 2.1%-3.6% and 4.2%-7.3%, respectively. HORRAT values for the reproducibility showed 0.4-0.7, indicating acceptable precision between laboratories. The method was thus proposed to be used by the drug-abuse urine testing laboratories in Taiwan.

A collaborative study was performed to assess accuracy, repeatability, and reproducibility of a nearinfrared (near-IR) method for determining crude protein content (PC) of whole-grain wheat. Four types of commercially available near-IR instruments, representing various combinations of wavelength region, mode of energy capture, method of energy dispersion, and treatment of spectral data, were used. Eight, 9,10, and 11 collaborators were involved, the exact number depending on instrument type. All collaborators received 22 samples of whole-grain hard red winter (HRW) wheat. They were furnished reference PCs (i.e., protein concentrations, w/w) corrected to a 12% moisture basis for instrument standardization. AOAC Method 990.03— combustion analysis—was the reference procedure. Standardization consisted of performing one of the following treatments to the instrument manufacturer's (or federal agency's) PC equation: (1) bias correction, (2) slope and intercept correction, or (3) recalibration with inclusion of standardization sample spectra. Standardized equations were then applied to a test set of 12 unknown HRW wheat sample spectra, with 2 samples blindly duplicated. The PCs of test samples ranged from 9 to 16%. Near-IR predictions were compared with reference measurements. Averaged within instrument type, root mean square of differences were 0.22, 0.24, 0.25, and 0.26% PC, depending on instrument. Corrected for bias within the test set, standard errors became 0.22, 0.18, 0.21, and 0.24% PC, respectively. These values were approximately twice the estimated lower limit for error (representing sample inhomogeneity). Overall repeatability relative standard deviation (RSD,) values were 0.92, 0.36, 0.42, and 0.74%, respectively. Overall reproducibility relative standard deviation (RSDR) values were 1.15, 0.61,1.53, and 1.38%. Such values for within-laboratory and between-laboratory variations of the near- IR methods were equivalent to values reported for the combustion method (990.03) for wheat. An inhouse study that examined all 6 U.S. wheat classes with one of the 4 instrument types produced repeatability and reproducibility values similar to those of the collaborative study, suggesting that the near-IR technique may be applied to red, white, hard, soft, and durum wheats. The near-IR method for determination of PC of whole-grain wheat has been adopted First Action (997.06) by AOAC INTERNATIONAL.

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.

Results are presented from an NMKL (Nordic Committee on Food Analysis) collaborative study of a method for the determination of cholecalciferol (vitamin D3) in foods. The method is based on the addition of an internal standard (vitamin D2), followed by saponification and extraction with n-heptane. The fraction that contains vitamin D2/D3 is separated by preparative normal-phase liquid chromatography (LC), and the analytes are determined by reversed-phase LC with UV detection at 265 nm. The method was tested by 8 participating laboratories. In this study 6 different matrixes were analyzed for cholecalciferol content: milk, liquid infant formula (gruel), cooking oil, margarine, infant formula, and fish oil. The contents varied from 0.4 to 12 microg/100 g. Three matrixes (milk, gruel, and margarine) were fortified with vitamin D3. In the other matrixes, vitamin D3 was added at 3 different levels at the Swedish National Food Administration. The milk was analyzed as a blind duplicate, whereas the other matrixes were analyzed as split-level pairs. The recoveries from the samples with vitamin D3 added varied from 93 to 102%. The repeatability relative standard deviation (RSDr) values for accepted results varied between 2.2% (fish oil) and 7.4% (cooking oil), whereas the reproducibility relative standard deviation (RSD(R)) values varied between 6.8% (margarine) and 24% (cooking oil).

An international collaborative study was conducted of a high-performance liquid chromatography (HPLC)-UV method for the determination of the major (ephedrine [EP] and pseudoephedrine [PS]) and minor (norephedrine [NE], norpseudoephedrine [NP], methylephedrine [ME], and methylpseudoephedrine [MP]) alkaloids in selected dietary supplements representative of the commercially available products. Ten collaborating laboratories determined the ephedrine-type alkaloid content in 8 blind replicate samples. Five products contained ephedra ground herb or ephedra extract. These 5 products included ground botanical raw material of Ephedra sinica, a common powdered extract of Ephedra sinica, a finished product containing only Ephedra sinica ground botanical raw material, a complex multicomponent dietary supplement containing Ma Huang, and a high-protein chocolate flavored drink mix containing Ma Huang extract. In addition, collaborating laboratories received a negative control and negative control spiked with ephedrine alkaloids at high and low levels for recovery studies. Test extracts were treated to solid-phase extraction using a strong-cation exchange column to help remove interferences. The HPLC analyses were performed on a polar-embedded phenyl column using UV detection at 210 nm. Repeatability relative standard deviations (RSDr) ranged from 0.64-3.0% for EP and 2.0-6.6% for PS, excluding the high protein drink mix. Reproducibility relative standard deviations (RSDR) ranged from 2.1-6.6% for EP and 9.0-11.4% for PS, excluding the high protein drink mix. Recoveries ranged from 84.7-87.2% for EP and 84.6-98.2% for PS. The data developed for the minor alkaloids are more variable with generally unsatisfactory HORRATS (i.e., >2). However, since these alkaloids generally add little to the total alkaloid content of the products, the method gives satisfactory results in measuring total alkaloid content (RSDr 0.85-3.13%; RSDR 2.03-10.97%, HORRAT 0.69-3.23, exclusive of the results from the high protein drink). On the basis of these results, the method is recommended for Official First Action for determination of EP and PS in dietary supplements exclusive of the high protein drinks.

Interlaboratory performance statistics was determined for a method developed to measure the resistant starch (RS) content of selected plant food products and a range of commercial starch samples. Food materials examined contained RS (cooked kidney beans, green banana, and corn flakes) and commercial starches, most of which naturally contain, or were processed to yield, elevated RS levels. The method evaluated was optimized to yield RS values in agreement with those reported for in vivo studies. Thirty-seven laboratories tested 8 pairs of blind duplicate starch or plant material samples with RS values between 0.6 (regular maize starch) and 64% (fresh weight basis). For matrixes excluding regular maize starch, repeatability relative standard deviation (RSDr) values ranged from 1.97 to 4.2%, and reproducibility relative standard deviation (RSDR) values ranged from 4.58 to 10.9%. The range of applicability of the test is 2-64% RS. The method is not suitable for products with <1% RS (e.g., regular maize starch; 0.6% RS). For such products, RSDr and RSDR values are unacceptably high.

An attenuated total reflection infrared spectroscopy procedure was collaboratively studied among two sets of five laboratories for quantitating the total trans fatty acid levels in neat (without solvent) hydrogenated vegetable oils, measured as triacylglycerols in one study, and as fatty acid methyl ester derivatives in the other. Unlike the fatty acid methyl esters, the triacylglycerols required no derivatization but had to be melted prior to measurement. To obtain a symmetric absorption band at 966 cm−1 on a horizontal background, the single‐beam spectrum of the trans‐containing fat was “ratioed” against that of a refined oil or a reference material that contained only cis double bonds. A single‐bounce horizontal attenuated total reflection cell that requires 50 µL of undiluted test samples was used for oils, melted fats, or their methyl esters. For fatty acid methyl esters, the reproducibility relative standard deviations were in the range of 0.9 to 18.46% for 39.08 to 3.41% trans, determined as methyl elaidate per total fatty acid methyl esters. For five pairs of triacylglycerol blind duplicates, the reproducibility and repeatability relative standard deviations were in the ranges of 1.62 to 18.97%, and 1.52 to 13.26%, respectively, for 39.12 to 1.95% trans, determined as trielaidin per total triacylglycerols. Six pairs of spiked triacylglycerol blind duplicates (quality assurance standards) exhibited high accuracy in the range of 0.53 to 40.69% trans and averaged a low bias of 1.3%. These statistical analysis results were compared to those collaboratively obtained by the recently adopted AOCS Cd14‐95 and AOAC 994.34 Infrared Official Methods.

This collaborative study was conducted to determine the total monomeric anthocyanin concentration by the pH differential method, which is a rapid and simple spectrophotometric method based on the anthocyanin structural transformation that occurs with a change in pH (colored at pH 1.0 and colorless at pH 4.5). Eleven collaborators representing commercial laboratories, academic institutions, and government laboratories participated. Seven Youden pair materials representing fruit juices, beverages, natural colorants, and wines were tested. The repeatability relative standard deviation (RSDr) varied from 1.06 to 4.16%. The reproducibility relative standard deviation (RSDR) ranged from 2.69 to 10.12%. The HorRat values were < or = 1.33 for all materials. The Study Director recommends that the method be adopted Official First Action.

Thirteen laboratories participated in an interlaboratory method performance (collaborative) study on a method for the determination of arsenic, cadmium, mercury, and lead by inductively coupled plasma/mass spectrometry (ICP/MS) after pressure digestion including the microwave heating technique. Prior to the study, the laboratories were able to practice on samples with defined element levels (pretrial test). The method was tested on a total of 7 foodstuffs: carrot puree, fish muscle, mushroom, graham flour, simulated diet, scampi, and mussel powder. The elemental concentrations in mg/kg dry matter (dm) ranged from 0.06-21.4 for As, 0.03-28.3 for Cd, 0.04-0.6 for Hg, and 0.01-2.4 for Pb. The materials used in the study were presented to the participants as blind duplicates, and the participants were asked to perform single determinations on each sample. The repeatability relative standard deviations (RSDr) for As ranged from 3.8 to 24%, for Cd from 2.6 to 6.9%, for Hg from 4.8 to 8.3%, and for Pb from 2.9 to 27%. The reproducibility relative standard deviations (RSDR) for As ranged from 9.0 to 28%, for Cd from 2.8 to 18%, for Hg from 9.9 to 24%, and for Pb from 8.0 to 50%. The HorRat values were less than 1.5 for all test samples, except for the determination of Pb in wheat flour at a level close to the limit of quantitation (0.01 mg/kg dm). The study showed that the ICP/MS method is satisfactory as a standard method for elemental determinations in foodstuffs.

ABSTRACTObjective:The aim of this study was to determine the kinetics of true ileal protein digestion and digestible indispensable amino acid score (DIAAS) of a goat milk-based infant formula (GIF), a cow milk-based infant formula (CIF), and human milk (HM).Methods:The GIF, CIF, and HM were investigated in an in vitro gastrointestinal model simulating infant conditions. Digested compounds were dialyzed from the intestinal compartment as bioaccessible fraction. Dialysate was collected in 15 to 60-minute periods for 4 hours. True ileal protein digestibility and DIAAS were determined as bioaccessible nitrogen (N) and amino acids.Results:N bioaccessibility from the GIF showed similar kinetics to that of HM. The CIF showed a delay in N bioaccessibility versus the GIF and HM. In the 1st hour of digestion, N bioaccessibility was 19.9% ± 3.5% and 23.3% ± 1.3% for the GIF and HM, respectively, and 11.2% ± 0.6% for CIF (P < 0.05 vs HM). In the 3rd hour of digestion, the N bioaccessibility was higher (P < 0.05) for the CIF (28.9% ± 1.2%) than for the GIF (22.5% ± 1.6%) and HM (20.6% ± 1.0%). After 4 hours, the true ileal protein digestibility of the GIF, CIF, and HM was 78.3% ± 3.7%, 73.4% ± 2.7%, and 77.9% ± 4.1%, respectively. The DIAAS for the GIF, CIF, and HM for 0- to 6-month-old infants was 83%, 75%, and 77% for aromatic AA.Conclusion:The protein quality is not different between the GIF, CIF, and HM, but the kinetics of protein digestion of the GIF is more comparable to that of HM than that of the CIF.

Simple SummaryIn fish farming, protein is the most expensive macronutrient in the diet. Research on the physiology of fish nutrition has focused on the relationships between nutrient intake and animal development and metabolism. The dynamics of protein metabolism are influenced by the body’s nutrient needs and dietary protein and energy intake. The Amazonian Pintado, a crossbred hybrid of Pseudoplatystoma reticulatum × Leiarius marmoratus, is farmed due to its growth rate, feed conversion efficiency, carcass yield, and absence of intramuscular bones. This study aimed to evaluate the effects of dietary digestible protein levels on muscle growth dynamics and oxidative stress in the white muscle of Amazonian Pintado. The results revealed that high dietary levels of digestible protein, despite increasing protein and free amino acid concentrations in the muscle, restrict muscle hypertrophy and increase tissue exposure to oxidative damage in Amazonian Pintado farming.This study aimed to evaluate the effects of dietary digestible protein levels on the growth dynamics and oxidative stress status of white muscle fibers in Amazonian Pintado (Pseudoplatystoma reticulatum × Leiarius marmoratus). Four hundred and fifty-five juveniles of Amazonian Pintado were fed diets containing varying digestible protein levels (225, 250, 275, 300, 325, 350, or 375 g kg−1) for 75 days. At the end of the experiment, the fish were fasted for 24 h, anesthetized, and euthanized to obtain muscle samples. The linear and quadratic effects of dietary digestible protein levels on white muscle fiber diameter, metabolite concentrations, and oxidative stress were assessed. The results revealed that increasing dietary digestible protein levels linearly raised the concentrations of free amino acids and total proteins in muscle tissue but also led to elevated levels of TBARS, indicating increased oxidative stress. Notably, the average area of muscle fibers with a cell area greater than 1133 µm2 decreased, reflecting restricted muscle hypertrophy, whereas glycogen and glucose levels also declined. These findings suggest that although high dietary digestible protein enhances protein and free amino acid concentrations in muscle tissue, it may compromise muscle hypertrophy and increase oxidative damage in Amazonian Pintado, underscoring the complexity of optimizing diet formulation.

Summary Hulls from Tower rapeseed (dark colored cultivar), R500 rapeseed (yellow colored cultivar) and soybean were included in semipurified diets for growing pigs at 10%, to investigate the effects of these fiber sources on dry matter, energy and protein digestibilities. Celufil, a purified fiber source, was also included in another diet at a 10% level for comparison. Casein, which was the main protein source, was added at a 15% level in all the diets. Four 40-kg barrows fitted with cannulas at the terminal ileum were used in a digestibility trial with a 4  4 Latin square design. The digestibilities of dry matter, protein and amino acids were measured to the ends of both the small and large intestines, while the digestibility of energy was measured only over the entire gastrointestinal tract. The true protein digestibility was also determined at the ileal and fecal levels. The Celufil diet had a higher cellulose content (8.7%) than the other diets (2.8 to 5%). The