Single-Laboratory Validation of AOAC Official Method 2011.10 for Vitamin B12 in ‘Indian’ Infant and Pediatric Formulas and Adult Nutritionals

Background: Ensuring the quality of infant and pediatric formulas and adult nutritionals is of utmost importance for the health and safety of rapidly urbanizing Indian population. B12 is an important water-soluble vitamin, which is fortified externally in such nutritional formulations. The Bureau of Indian Standards (BIS) has a recommended microbiological assay–based method for determination of vitamin B12 that is not precise and accurate enough to meet the label claim requirements of infant, adult, and/or pediatric nutritionals. The AOAC Official Method 2011.10 was originally developed under the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) for the determination of vitamin B12 in infant and pediatric formulas and adult nutritionals. However, those SPIFAN matrixes did not contain malt and other indigenous cereal and legume flour (with or without cocoa powder), which are commonly found in Indian formulations. Thus, there is a need to replace this method with a more precise and accurate method. Objective: This study was undertaken to validate the AOAC Official Method 2011.10 on vitamin B12 in ‘Indian’ infant and pediatric formulas and adult nutritionals. Methods: The single-laboratory validation (SLV) of AOAC Method 2011.10 was carried out as per the AOAC Guidelines in six Indian pediatric and adult nutritional formulas to verify its fitness for purpose. Cobalamin in the sample was converted to cyanocobalamin on treatment with potassium cyanide. The sample was then subjected to clean up through a C18 cartridge. Vitamin B12 in the eluted extract was separated from other components using size-exclusion column chromatography followed by a C18 column. The HPLC analysis was carried out at 550 nm. Results: Diastase treatment and C18 solid-phase extraction cleanup satisfactorily removed the matrix interference. The relative standard deviation of the determined values in 30 samples each from 6 selected Indian products and NIST SRM 1849a was <20%. The average recoveries for the spiked recovery samples ranged from 91.75 to 101.14%. Conclusions: Method 2011.10 met the standard method performance requirements set forth by the AOAC SPIFAN. Therefore, we recommend the Method 2011.10 for adoption as the BIS official method for the analysis of vitamin B12 in ‘Indian’ infant and pediatric formulas and adult nutritionals. Highlights: This was the first SLV project that the AOAC India section undertook to extend the scope of the AOAC Method 2011.10 for vitamin B12 analysis by validating it in ‘Indian’ infant and pediatric formulas and adult nutritionals.

Improvement in the Reliability of AOAC Official Method 2012.15 for Iodine

Background: Reliable measurements of iodine are essential for ensuring the qualityof infant formula. The AOAC Official Method 2012.15 for iodine tends to produce higher results in the presence of carbon remaining in the final test solution after digestion with alkaline dissolution. This is partly because of the lack of countermeasures for signal enhancement induced by coexisting carbon in Method 2012.15. Objective: To obtain more reliable values for infant formulas, we undertook an experiment. Methods: We modified the protocol by addingcarbon in the form of methanol to both the standardsolutions and the final test solutions. Comparisonsof the enhancement factor for iodine-127 were usedto find the optimized concentration of methanol from0–10%. Results: Optimization of the additional carbon showed that a 5% methanol minimum was necessary for a constant ratio of iodine. The results exhibited good linearity (coefficient of determination >0.999), and the LOQ was 0.19 μg/100 g for the reconstituted final product with a methanol concentration of 5%. The intermediate precision RSD was <3.76%, and the recovery factor was 97.5–104.2% for infant formula distributed in several countries and a special formula distributed in Japan. Conclusions: This demonstrates that 5% methanol, when addedto standard and final solutions, acts as an effective matrix matching agent. Highlights: This modified method produces more accurate iodine quantification in infant formulas and special formulas in which there is incomplete digestion of the matrix.

KANDUNGAN AFLATOKSIN PADA LADA INDONESIA DALAM PENGEMBANGAN STANDAR INTERNASIONAL CODEX

Lada merupakan salah satu rempah-rempah yang dihasilkan dari sektor perkebunan. Komoditas lada Indonesia juga diekspor ke negara lain. Adanya kandungan aflatoksin dalam rempah-rempah saat ini menjadi isu hangat dalam sidang Codex. Penelitian ini bertujuan untuk mengetahui kandungan aflatoksin pada lada dalam rangka mendukung pengembangan standar Codex. Sampel lada yang diambil yaitu biji lada yang berasal dari petani, pengepul dan eksportir. Lokus pengambilan sampel dari Lampung, Bangka dan Kutai. Jumlah total sampel dalam penelitian ini sebanyak 26 sampel lada. Pengujian terhadap sampel lada yang telah diambil dilakukan di laboratorium yang telah bekerjasama yaitu Balai Pengujian Mutu Barang-Kementerian Perdagangan. Dalam analisis data kandungan aflatoksin pada lada, metode pengujian yang digunakan adalah metode AOAC Official Method 991.31.2005 dengan LOQ berturut-turut 1,07 ppb (Aflatoksin B1); 0,39 ppb (Aflatoksin B2); 1,35 ppb (Aflatoksin G1), dan 0,48 ppb (Aflatoksin G2). Hasil penelitian menunjukkan bahwa seluruh sampel lada nilai kandungan aflatoxin B1 < LOQ, sedangkan kandungan total aflatoxin juga < LOQ. Nilai kandungan tersebut masih dikategorikan aman untuk dikonsumsi karena dibawah ambang batas yang ditetapkan BPOM dan regulasi Uni Eropa.

Improvement of Versatility and Analytical Range of AOAC Official Method 2015.06 for Selenium

AOAC Official Method 2015.06 is not applicable for infant formula without selenium addition because of lack of sensitivity. In addition, Method 2015.06 specifies hydrogen gas as the cell gas of inductively coupled plasma (ICP)-MS instruments. There are only a few manufacturers who have formally adopted hydrogen gas. To expand the applicability of Method 2015.06 for infant formulas with lower selenium content and for ICP-MS instruments that do not use hydrogen gas as the cell gas, we modified the conditions of Method 2015.06. The results exhibited a good linearity (coefficient of determination >0.999) when the range of standard concentration was set from 0.4 to 16.0 μg/L and the cell gas was replaced with helium gas. The measurement precision was improved to an intermediate precision RSD value of 3.49%, and the recovery factor was 103.1%. This study demonstrates that helium gas can be used as the cell gas (easing restrictions in selecting an ICP-MS instrument) and expands the applicability of this method to infant formula samples with lower selenium content by modifying the sample preparation method.

Modification and Single-Laboratory Validation of AOAC Official Method 977.13 for Histamine in Seafood to Improve Sample Throughput

Histamine is the main causative agent in scombrotoxin fish poisoning, the most frequently reported illness related to fish consumption. The AOAC official method for histamine determination in fish is the fluorometric method AOAC 977.13, which is sensitive and reproducible but somewhat labor intensive and time consuming. We investigated multiple modifications to this method in an attempt to reduce assay time and increase sample throughput while maintaining the performance of the original method. Some of the attempted modifications negatively affected the performance characteristics of the method. However, omitting the heating step during extraction and replacing the cuvette style fluorometer with a microplate reader retained method performance while increasing sample throughput. Therefore, we adopted these modifications and conducted a single-laboratory validation. The recovery, precision (RSD), and LOD of the modified method assessed by the single-laboratory validation ranged from 92 to 105%, 1 to 3%, and 0.2 to 0.5 ppm, respectively, in tuna, mahi-mahi, and Spanish mackerel samples. We conclude that the AOAC 977.13 fluorometric method, modified as described, will improve assay time and sample throughput efficiency cumulatively, as the number of sample units analyzed increases. We anticipate that this modified method could be used by regulatory agencies and other laboratories following successful multilaboratory validation.

Akumulasi dan Depurasi Toksin PSP (Paralytic Shellfish Poisoning) oleh Kerang Hijau (Accumulation and Depuration of PSP Toxin (Paralytic Shellfish Poisoning) by Green Mussels)

Ledakan mikroalga sering dilaporkan terjadi di Teluk Jakarta, dimana di lokasi tersebut juga terdapat kegiatan budidaya kerang hijau (Perna viridis). Terkait dengan hal tersebut maka dilakukan studi akumulasi dan depurasi toksin PSP (Paralytic Shellfish Poisoning) pada kerang hijau. Studi akumulasi dilakukan di bagan kerang hijau perairan Cilincing Jakarta Utara, dengan memisahkan kerang hijau yang berukuran sama dan ditempatkan kembali ke bagan. Sampling dilakukan setiap minggu selama 2 bulan dan diukur juga kelimpahan fitoplankton, pH, suhu dan salinitas perairan. Depurasi dilakukan di Unit Depurasi Kekerangan KKP Panimbang Banten, yang dilakukan selama 24 jam. Pencuplikan sampel dilakukan setiap jam pada 4 jam pertama dan setiap 2 dan 3 jam pada waktu berikutnya. Penentuan konsentrasi toksin PSP dilakukan dengan menggunakan HPLC detektor fluoresensi. Prosedur preparasi, ekstraksi dan pengukuran konsentrasi toksin mengikuti Manual AOAC Official Method 2005.06 untuk toksin PSP dalam kekerangan. Akumulasi toksin PSP oleh kerang hijau di perairan Cilincing pada bulan Januari–Pebruari 2011 berkisar antara 4,11–11,96 µg STX eq. per 100 g dan tidak mempunyai korelasi dengan kelimpahan Dinoflagelata di perairan. Hal ini disebabkan uji akumulasi tidak dilakukan pada saat blooming mikroalga. Uji depurasi selama 24 jam mengeliminasi toksin PSP sebesar 60%, sehingga bisa diajukan sebagai sistem pemutus rantai toksin dari mikroalga ke manusia. Kata kunci: akumulasi, depurasi, PSP toksin, kerang hijau, Cilincing Microalgae blooms have been frequently reported in the Jakarta Bay, which is also the location of green mussel (Perna viridis) aquaculture. Accumulation and depuration of Paralytic Shellfish Poisoning (PSP) toxin in the green mussels were investigated in the field, where the toxin accumulation studies conducted in the mussel farming at Cilincing, North Jakarta. Accumulation test carried out by placing back the selected green mussel (equal size) into the mussel farming. Every week for 2 months, the green mussel were collected from mussel farming and transported to the laboratory. The fitoplankton abundance also was checked including pH, Suhue and salinitiy paramaters. Toxin depuration was conducted at Clams Sanitation Unit at Panimbang Banten. The depuration studies were conducted for 24 hours with sampling every hour in the first 4 hours and every 3 and 2 hours until the 24th hour. Preparation, extraction and toxin concentration measurements performed by following the Manual AOAC Official Method 2005.06 for PSP toxin in oyster. This research concluded that the accumulation of PSP toxin by green mussel, Perna viridis in the mussel farming at Cilincing, North Jakarta in ranged between 4,11–11,96 µg STX eq. per 100 g during January–February 2011. No correlation between PSP toxin concentration in the green mussel, Perna viridis with abundance of the PSP toxin sources phytoplankton, because the study wasnt done when microalgae blooming. The depuration processes was eliminate 60% the PSP toxins for 24 hours depuration processing. It can be proposes as a banded system the PSP toxin from algae to human being. Keywords: accumulation, depuration, PSP toxin, green mussel, Cilincing

Interlaboratory Comparison of Two AOAC Liquid Chromatographic Fluorescence Detection Methods for Paralytic Shellfish Toxin Analysis through Characterization of an Oyster Reference Material

An interlaboratory ring trial was designed and conducted by the Centre for Environment, Fisheries, and Aquaculture Science to investigate a range of issues affecting the analysis of a candidate Pacific oyster paralytic shellfish toxin reference material. A total of 21 laboratories participated in the study and supplied results using one or more of three instrumental methods, specifically precolumn oxidation (Pre-COX) LC with fluorescence detection (FLD; AOAC Official Method 2005.06), postcolumn oxidation (PCOX) LC-FLD (AOAC Official Method 2011.02), and hydrophilic interaction LC/MS/MS. Each participant analyzed nine replicate samples of the oyster tissue in three separate batches of three samples over a period of time longer than 1 week. Results were reported in a standardized format, reporting both individual toxin concentrations andtotal sample toxicity. Data were assessed to determine the equivalency of the two AOAC LC methods and the LC/MS/MS method as well as an assessment of intrabatch and interbatch repeatability and interlaboratory reproducibility of each method. Differences among the results reported using the three methods were shown to be statistically significant, although visualcomparisons showed an overlap between results generated by the majority of tests, the exception being the Pre-COX quantitation of N-hydroxylated toxins in post ion-exchange fractions. Intralaboratory repeatability and interlaboratory reproducibility were acceptable for most of the results, withthe exception of results generated from fractions. The results provided good evidence for the acceptableperformance of the PCOX method for the quantitation of C toxins. Overall the study showed the usefulnessof interlaboratory analysis for the characterizationof paralytic shellfish poisoning matrix reference materials, highlighting some issues that may need to be addressed with further method assessment at individual participant laboratories.

Determination ofβ-Galactooligosaccharides by Liquid Chromatography

Beta-galactooligosaccharides (GOS) are oligosaccharides normally produced industrially by transgalactosylation of lactose. They are also present naturally in the milk of many animals including humans and cows. GOS are thought to be good for health, being potential prebiotic fibres, and are increasingly added to food products. In order to control the GOS content of products, the AOAC official method 2001.02 was developed. However, the method has some shortcomings and in particular is unsuited to the analysis of products containing high levels of lactose such as infant formula. To overcome this problem, we developed a new method for application to infant formula and tested it on various GOS ingredients as well as infant formulae. When applied to GOS ingredients the results of the new method compare well with those of the official AOAC method, typically giving results in the range 90–110% of those of the official method and having an expanded measurement uncertainty of less than 15%. For three products, the results were outside this range (recoveries of 80–120% and expended measurement uncertainties up to 20%). When applied to the analysis of infant formula, recoveries were in the range of 92–102% and the expanded measurement uncertainties were between 4.2 and 11%.

Modifications of AOAC Official Method 999.15 to Improve the Quantitation of Vitamin K1 in Complex Formulated Nutritional Products

Vitamin K1 (phylloquinone) occurs in foods in relatively low concentrations. It is synthesized for addition to formulated nutritional products (infant formulas, medical foods, and adult nutritional products). In recent years, nutritional products formulated with free amino acids and partially hydrolyzed proteins have been introduced in the market. Schimpf et al. demonstrated that the current AOAC Official Method 999.15 for determination of vitamin K in milk and infant formula is not adequate to quantitatively extract vitamin K1 from such products. We developed a modification of AOAC 999.15 for the analysis of vitamin K1 in these products that provides quantitative extraction by increasing the sample size, volume of extraction solvents, time of liquid/liquid partitioning, and order of the addition of solvents. This modified procedure showed extraction efficiency comparable to that of the original AOAC 999.15 procedure for analyzing infant formula matrixes and to the modified procedure of Schimpf et al. for the analysis of samples containing limited amounts of free amino acids and/or partially hydrolyzed proteins. Extraction efficiency increased more than 10% using the modified extraction procedure for samples containing higher amounts of these components. The chromatographic separation was improved by using a Dionex Acclaim triacontanol-bonded C30 column (250 × 3.0 mm id, 3 μm particle size) maintained at 15°C, with acetonitrile–methanol (50 + 50, v/v) mobile phase at a flow rate of 0.5 mL/min, which provided baseline separation of the cis and trans isomers of vitamin K1 from each other and from other compounds contained in the sample extracts.