Immunoassays and Mass Spectrometry for Determination of Protein Concentrations in Genetically Modified Crops.

Quantifying protein levels in genetically modified (GM) crops is crucial in every phase of development, deregulation, and seed production. Immunoassays, particularly enzyme-linked immunosorbent assay, have been the primary protein quantitation techniques for decades within the industry due to their efficiency, adaptability, and credibility. Newer immunoassay technologies like Meso Scale Discovery and Luminex offer enhanced sensitivity and multiplexing capabilities. While mass spectrometry (MS) has been widely used for small molecules and protein detection in the pharmaceutical and agricultural industries (e.g., biomarkers, endogenous allergens), its use in quantifying protein levels in GM crops has been limited. However, as trait portfolios for GM crop have expanded, MS has been increasingly adopted due to its comparable sensitivity, increased specificity, and multiplexing capabilities. This review contrasts the benefits and limitations of immunoassays and MS technologies for protein measurement in GM crops, considering factors such as cost, convenience, and specific analytical needs. Ultimately, both techniques are suitable for assessing protein concentrations in GM crops, with MS offering complementary capabilities to immunoassays. This comparison aims to provide insights into selecting between these techniques based on the user's end point needs.

Determination of Free and Total Choline and Free and Total Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula by Liquid Chromatography/Tandem Mass Spectrometry (HPLC-MS/MS): A Multi-laboratory Testing Study, Final Action 2015.10.

BACKGROUND: A multi-laboratory study was conducted on AOAC First Action Method 2015.10 "Determination of Free and Total Choline and Free and Total Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula by Liquid Chromatography/Tandem Mass Spectrometry (HPLC-MS/MS)." OBJECTIVE: In this study, nine laboratories participated in the performance testing of the method using ten nutritional products tested as blind duplicates. METHOD: Both free and total carnitine and free and total choline content of the samples were determined using separate extractions for the free and total results. For free choline and carnitine analysis, samples are diluted in water. For total choline and carnitine analysis, samples are extracted using acid-assisted microwave hydrolysis with nitric acid. For both the free and total methods, samples are then diluted with acetonitrile and analyzed using strong cation exchange (SCX) liquid chromatography coupled to a triple quadrupole tandem mass spectrometer (LCMS). Stable isotope labeled internal standards were utilized in all analyses to compensate for extraction inefficiencies and ionization suppression.

Determination of Bisphenol A (BPA) in Commercially Packaged Ready-to-Consume Carbonated and Noncarbonated Water and Nonalcoholic Beverages: A Single-Laboratory Validation Study, First Action 2017.15.

Background: Bisphenol A (BPA) is an endocrine-disrupting compound that could migrate to beverages from packaging materials. AOAC INTERNATIONAL issued a call for methods for determination of free BPA in beverages based on the AOAC Standard Method Performance Requirement (SMPR®) 2017.018. Objective: The objective of the single-laboratory validation (SLV) study was to validate a method for the analysis of BPA in relevant beverages and evaluate its performance characteristics against the AOAC SMPR 2017.18. Methods: The analytical method involves extraction of a beverage sample (10 mL) using 10 mL 1% acetic acid in acetonitrile after the addition of isotopically labeled internal standard. Sodium chloride is added to salt out BPA into the acetonitrile phase. After centrifugation, a freeze-out step is used to remove coextracted lipids. An aliquot of the supernatant upper layer is then analyzed using high-pressure LC coupled to tandem mass spectrometry in electrospray negative ionization mode. Results: Accuracy, repeatability, and intermediate precision were determined by spiking 10 representative nonalcoholic beverage matrixes at three concentration levels. Individual percent recoveries ranged between 76.5 and 113.2%, 86.2 and 114.8%, and 90.9 and 109.3% for the spike levels of 0.3, 1.5, and 20 µg/L, respectively. The repeatability and intermediate precision results were in the range of 0.2-11.3% and 1.8-11.1%, respectively. The LOD was estimated at ≤0.1 µg/L and LOQ was validated at 0.3 µg/L. Conclusions: The validated method performance characteristics met the requirements stated in the AOAC SMPR 2017.018. This method was approved AOAC First Action Official MethodsSM 2017.15. Highlights: SLV study evaluating free BPA at three concentration levels (0.3, 1.5, and 20 µg/L) in 10 types of commercially packaged nonalcoholic beverages (as recommended for the method validation in the AOAC SMPR 2017.018). Validated method performance characteristics (LOD, LOQ, analytical range, accuracy, and precision) met the AOAC SMPR 2017.018. Method approved as the AOAC First Action Official Method 2017.15.

Analytical Method for Lactoferrin in Milk-Based Infant Formulas by Signature Peptide Quantification with Ultra-High Performance LC-Tandem Mass Spectrometry.

Background: There is a need for a standardized method for quantification of lactoferrin in infant formulas, and manufacturers have started fortifying lactoferrin to mimic the higher levels found in human milk. A variety of current methods exist, but specificity and accuracy are challenging with the infant formula matrix. The use of signature peptides and MS is becoming more prevalent in the realm of analytical chemistry for quantification of proteins. Objective: The objective of this work was to develop and validate a method through a single-laboratory validation for quantification of lactoferrin in milk-based infant formula and begin to lay the foundation for a standardized method. Methods: The method presented uses signature peptides to quantify lactoferrin in milk-based infant formulas by ultra-high performance LC-tandem mass spectrometry (MS/MS). These peptides are produced through tryptic digestion, and fragments produced from these peptides through MS/MS allow the specific quantification using correlating isotopically labeled peptides. Results: The validation parameters were all met with precision RSDr ranging from 2.1 to 7.1 and intermediate RSDR ranging from 7.0 to 10.4 across different fortified milk-based infant formulas. Accuracy with certified reference material resulted in mean recoveries of 91.7-96.4%. Conclusions: The results from this study demonstrate the method is fit for purpose to support manufacturing specifications and nutritional labeling requirements.

Method development and validation for low-level propineb and propylenethiourea analysis in baby food, infant formula and related matrices using liquid chromatography-tandem mass spectrometry.

Two simple, selective and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated for determination of propineb and propylenethiourea (PTU) in infant formula, fruit-based and cereal-based baby food and raw materials used in production of infant formula, including carbohydrates, protein isolates, vegetable oils and emulsifiers. The sample preparation procedure for propineb analysis was based on streamlined derivatisation to form and stabilise the target analyte (propylenebisdithiocarbamate-dimethyl), followed by extraction using a modified QuEChERS procedure with a dispersive solid phase extraction (d-SPE). The PTU determination employed an aqueous extraction with optimised protein precipitation and single-step SPE clean-up. To achieve maximum sensitivity, electrospray ionisation and atmospheric-pressure chemical ionisation were employed for LC-MS/MS analysis of propineb and PTU, respectively. Validation of the developed methods was performed in accordance with Document SANTE/11813/2017. Mean recoveries were in the range of 86-120% for propineb and PTU, respectively, with interday and intraday repeatabilities below 13%. A limit of quantification (LOQ) of 0.003 mg kg-1 was validated for most of the evaluated analyte/sample matrix combinations with the exception of PTU in soy protein isolate and soybean oil, for which an LOQ of 0.01 mg kg-1 was obtained. This is the first report that provides validated methods for monitoring propineb and PTU in infant formula and baby foods at concentrations compliant with the maximum residue levels established in the EU legislation.

Determination of Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS): Single-Laboratory Validation, First Action 2015.10.

Analytical methods for the analysis of both L-carnitine and choline are needed for reliable and accurate determination in infant formula and adult/pediatric nutritional formula. These compounds are different in how they are utilized by the human body, but are structurally similar. L-carnitine and choline are quaternary ammonium compounds, enabling both to be retained under acidic conditions with strong cation exchange (SCX) chromatography. This method analyzes both compounds simultaneously as either the free forms or as a total amount that includes bound sources such as phosphatidylcholine or acetylcarnitine. The free analysis consists of water extraction and analysis by LC/MS/MS, while the total analysis consists of extraction by acid assisted microwave hydrolysis and analysis by LC/MS/MS. Calibration standards used for calculations are extracted with all samples in the batch. A single laboratory validation (SLV) was performed following the guidelines of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) utilizing the kit of materials provided. The results achieved meet the requirements of SMPR 2012.010 and 2012.013 for L-carnitine and total choline, respectively.