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Determination of Casein Allergens in Extensively Hydrolyzed Casein Infant Formula by Liquid Chromatography-Tandem Mass Spectrometry

Abstract Background The use of hypoallergenic infant formulas and the need for reliable tests to determine the presence of residual antigens have increased in parallel. Objective An LC-MS method for quantitation of casein was validated using incurred samples and a matrix-matched external standard curve. Method Powdered infant formula samples were extracted in a buffer of sodium deoxycholate and ammonium bicarbonate at 60°C and filtered through 7 kDa desalting columns. Samples were digested overnight with trypsin and precipitated with acid prior to analysis of marker peptides by tandem mass spectrometry. Results Based on three marker peptides, the linear range for casein was 1.8–42 μg/g of powdered infant formula with an LOQ of 1.8 μg/g. The determination coefficients (R2) for each curve were ≥0.99 for casein peptides. Method repeatability was ≤22% RSD and intermediate precision was ≤23% RSD; recovery of casein from incurred material (2–20 µg/g) ranged from 78% to 118%. Conclusions An LC-MS/MS method was developed and validated for confirmation of casein allergens in hypoallergenic infant formula. Highlights A method was developed to accurately and reliably quantify casein allergens in extensively hydrolyzed casein infant formula by LC-MS without the need for custom peptide standards.

case of formulas made with extensively hydrolyzed proteins such as casein, allergen testing also provides assurance that no intact protein contaminates either ingredients or the final product. Several analytical methods have been used to measure allergens in extensively hydrolyzed casein infant formula, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), and immunoblotting (5)(6)(7). Of these, SDS-PAGE and immunoblotting are more time-consuming and are primarily used for research and validation purposes, while ELISA is more often used for quality control. Although an SDS-capillary gel electrophoresis method for whey protein in infant formula was validated, it does not measure casein and is not applicable to hydrolyzed products (8).
Despite its widespread use for allergen testing, ELISA has several limitations when used for extensively hydrolyzed infant formula. For example, the use of polyclonal antibodies may overestimate allergens because of their different affinities for proteins and peptides (5). Antibodies raised against protein epitopes are also prone to cross reactivity with proteins of similar structure, and changes in protein conformation during food processing may influence results (9,10). Due to its specificity, LC-MS/MS has been proposed as a confirmatory test for allergens in a number of processed foods, including pasta, baked goods, baby food, breakfast cereal, and infant formula (9)(10)(11)(12)(13). Although two of these LC-MS/MS methods measured allergens in infant formula, neither were evaluated with hypoallergenic infant formula, which requires validation to ensure that hydrolyzed protein fragments do not interfere with the analysis.
To address the lack of specific confirmatory tests for routine use with hypoallergenic infant formula, we developed and validated extraction and LC-MS/MS procedures for determination of intact casein in an extensively hydrolyzed casein formula. Because intact casein contamination was possible from the casein ingredient as well as from milk in the manufacturing facility, the assay used tryptic peptides from the milk proteins a s1 -casein and a s2 -casein for quantification. The method was evaluated for precision, accuracy, and robustness under different assay conditions. While our laboratory has previously developed immunoassays for these and similar applications, our main purpose has been to design and implement tests that are not dependent on reagents that are not widely available for researchers and manufacturers of infant formulas.

Reagents
Note: Reagent volumes may be scaled up or down provided good laboratory practices are followed. (i) Extraction buffer (50 mM NH 4 HCO 3 , 1% SDC).-Add 3.6 g NH 4 HCO 3 and 900 mL water to a 2 L beaker and mix on a spin plate to dissolve. Add 9 g SDC and allow to slowly dissolve without stirring (approximately 30 min). When dissolved, mix briefly on a spin plate and transfer to a media bottle. This is enough solution for eight calibration solutions or samples.

Standards and In-House Reference Materials
The assay was tested using extensively hydrolyzed casein infant formula (Perrigo Nutritionals; Georgia VT, USA). This formula was manufactured by adding externally sourced extensively hydrolyzed casein to liquid ingredients prior to heat treatment and drying, and incurred sample was produced by adding nonfat dry milk (NFDM) to the same liquid ingredients during the manufacturing process. NFDM was used for the incurred sample because it is an ingredient for other formulas and is therefore a potential contaminant. Because of the known ion suppression of the sample matrix in LC-MS/MS methods, the untreated extensively hydrolyzed casein infant formula was used as the blank matrix. NFDM from Agri-Mark, Inc. (West Springfield, MA, USA) was used to make the in-house reference material for casein allergen and as a standard to quantify casein allergen in samples by spiking the blank matrix. The nitrogen concentration in the NFDM was determined by combustion using a LECO analyzer (LECO Corp., St. Joseph, MI, USA), and protein in the sample was calculated to be 35.3% using a nitrogen conversion factor of 6.38 (14). To calculate the casein concentrations in the standards and samples, it was assumed that casein accounted for 80% of protein (15). The incurred sample contained 20 mg/g casein (70.8 mg/g NFDM) on a powder basis, which is below the lowest observed adverse effect level (LOAEL) for milk allergens according the U.S. Food and Drug Administration (16) based on a 100 mL dose for infant formula. We estimated that a level of casein 10 ppm is clinically relevant based on LOAELs for whole milk protein in children (17) and results from a clinical trial confirming the hypoallergenicity of an extensively hydrolyzed casein formula (18). However, the method has been validated near the LOQ since clinically relevant targets may change as new information becomes available. For incurred samples at 2 and 10 mg/g, the inhouse reference material was diluted with the blank matrix at the time of sample preparation.

Preparation of Standard Solutions
Stock solution of NFDM (1 mg/mL) in water was prepared fresh daily. NFDM intermediate solution (100 mg/mL) was made by diluting NFDM stock solution with extraction buffer (50 mM NH 4 HCO 3 , 1% SDC).

Calibration Solutions
Protein extraction and trypsin digestion were done according to Lin et al. (19) and Lutter et al. (12) with modifications. A matrixmatched calibration curve was used to account for ion suppression in the sample matrix, a common challenge with processed foods (9,12,20). The calibration solutions were prepared alongside the test samples as described in the Sample Preparation section.

LC-MS Analysis
Quantitation by LC-MS/MS was done with an Agilent 1290 UHPLC system and Agilent 6460 triple quadrupole mass spectrometer using an ESI Jet Stream source and MassHunter software (Santa Clara, CA, USA). LC gradients were optimized with fused core columns using acidified water and acetonitrile mobile phases (11). A fused core XB-C18 analytical column (1.7 mm, 2.1 Â 150 mm) was connected to a matching guard column (Phenomenex, Torrance, CA) and the injection volume was 5 mL.
The column temperature was set to 60 C and the flow rate to 0.3 mL/min with the mobile phases 0.1% v/v formic acid aqueous (A) and acetonitrile (B). The gradient used for separation started with 97% aqueous formic acid and 3% acetonitrile changing linearly to 60 and 40%, respectively, in 10 min. The column was then washed for 2 min with 100% acetonitrile and reequilibrated in the initial eluent for 5 min. Peptides were quantified by MS in positive mode using the source parameters from Lutter et al. (12). The drying gas (N 2 ) and sheath gas temperatures were set at 350 C, drying gas and sheath gas flows at 10 L/min, nebulizing gas (N 2 ) pressure at 30 psi, capillary voltage at 3000 V, and nozzle voltage at 300 V. Precursor ions were based on marker peptides previously identified for casein, which were confirmed for specificity using BLAST searches (11,21). The marker peptides were further confirmed as selective for casein (as opposed to whey proteins such as a-lactalbumin or b-lactoglobulin) by spiking samples with whey protein standards. Product ions were based on those previously reported for the marker peptides, on in silico fragmentation using the Protein Prospector database (22), and on product ion scans from m/z 100 to 1400. Final multiple reaction monitoring (MRM) parameters are shown in Table 2.

Calculations
(a) Calculate the concentration of casein, in lg/mL, in each calibration solution:  Table 3.
where Cas NAVP ¼ the casein in the sample (lg/g powder), based on the marker peptide NAVPITPTLNR; A NAVP ¼ the peak area of the quantifier ion of the marker peptide in the sample chromatogram, in arbitrary units (AU); I NAVP ¼ the y-intercept of the calibration curve for the quantifier ion of the marker peptide; RF NAVP ¼ the slope of the calibration curve for the quantifier ion of the marker peptide; M S ¼ the sample weight, in g.

Method Validation
A single-laboratory validation was performed in accordance with ICH guidelines (23). Quantifying ions were used for calibration and quantitation, and specificity of the method was confirmed with qualifying ions for each marker peptide. To measure linearity and range, a 5-point standard curve for casein was made according to the method, and each standard was injected at the beginning and end of each chromatographic sequence. LOD was calculated as (3.3 Â s)/slope and LOQ was

Selectivity
Because allergen contamination could potentially occur at any point in the production process, it was necessary to demonstrate the ability to quantify intact proteins in a relevant incurred matrix. Extensively hydrolyzed casein infant formula incurred with NFDM was passed through 7 kDa MWCO desalting columns to ensure that only intact casein submitted to analysis. This proved effective, as marker peptides for casein were easily detected in incurred formula ( Figure 1) and no signal was seen in the blank sample matrix at the same retention time. Several marker peptides reported in the literature were initially tested, and those that showed the best selectivity were chosen for further method validation. Of the three eventually chosen to quantify intact casein in the samples, FFVAPFPEVFGK and YLGYLEQLLR from aS1-casein were reportedly found in IgE binding epitopes identified in sera of allergenic patients (24). Thus, in the case of these two peptides the test quantified potential contamination that is relevant from an immunological standpoint.
For LC-MS/MS analysis, the product ion with the greatest signal was used as the quantifying ion and the other product ions were used as qualifying ions. A total of four casein peptides were originally tested. Although measuring FALPQYLK from aS2-casein resulted in multiple peaks, the remaining three peptides showed good selectivity ( Figure 1) and were chosen for further validation. Relative intensity of qualifying ions in calibration solutions and samples is often used to confirm the presence of analytes by LC-MS/MS. Although criteria are not specified for relative ion intensity for peptide analysis, the requirements for identification of pesticide residues in foods based on relative ion intensity is outlined by the European Commission (25). To determine relative ion intensities (ion ratios), first ion ratios (quantifier ion peak area: qualifier ion peak area) were calculated for each calibration solution and incurred sample. The ratios of the samples were then compared with the average of the ion ratios of the calibration solutions in the same sequence. According to SANTE/11813/2017, the ion ratio of the samples should be within 30% (relative) of the average ion ratio of the calibration standards (25). All three casein peptides met these criteria (Table 3).

Linearity and range
Data for linearity and range from the 5-point calibration curve for casein are summarized in Table 4. The determination coefficients (R 2 ) for each curve were !0.99 for casein peptides. The range for casein in infant formula (approximately 1.8-42 lg/g) corresponds to approximately 6-150 lg/g NFDM in powdered infant formula and is a suitable range for hypoallergenic formula. This is similar to the range of 20-150 lg/g milk powder in soybased infant formula reported by Lutter et al., who used FALPQYLK from aS2-casein as a marker peptide (12). It is smaller than the range of 1-1000 lg/g whole milk in infant formula reported by New et al., who used YLGYLEQLLR from aS1-casein as a marker peptide (13).

LOD and LOQ
The calculated LOD and LOQ varied with the marker peptide used for measurement ( Table 4). Although these calculated values are lower than the range of the standard curve, it is recommended that the low end of the calibration range be used as the LOQ because the use of qualifier ions provides confidence in the results. The low LOQ with this method can likely be attributed to the desalting (size exclusion) step in the sample preparation. Size exclusion has been used in sample preparation for allergen detection in order to reduce ion suppression (9,13,26), but ideally smaller peptides would be included in the analysis. Casein fragments in the 3-6 kDa range have been shown to bind to human IgG antibodies (27), suggesting that our method cannot be expected to detect all immunogenic peptides.

Recovery and precision
Recovery of casein from the incurred infant formula samples ranged from 78 to 118%, varying with the peptide used for quantitation and the concentration of casein in the formula (Table 5). Previous studies have given various results. Lutter et al. were not able to recover b-, j-, or aS2-casein from soy-based infant formula spiked with 5-15 lg/g NFDM (12). New et al. spiked 10, 100, and 1000 lg/g whole milk powder into an infant formula and recovered 71-83% (13). AOAC INTERNATIONAL has published Standard Method Performance Requirements (SMPRV R ) for allergens by LC-MS (SMPR 2016.002) (28), and our method meets the requirement of 60-120% recovery. The NFDM used for the calibration curve in this study came from a local supplier, but it has nearly the same protein concentration as the MoniQA reference material for skim milk powder (35.3% versus 35.4%) (29) and would presumably produce similar results for quantifying milk in this assay. Repeatability and intermediate precision data are also shown in Table 5. With the exception of one peptide measured at 2 mg/g, the method meets the repeatability limit of 20% RSD specified in SMPR 2016.002. Although the SMPR does not specify criteria for intermediate precision, the maximum value for the method was 23% RSD iR .

Robustness
As stated above, we evaluated three different resins to desalt samples during the preparation process: polypropylene, polyacrylamide, and Sephadex G25 (dextran/epichlorohydrin). Samples prepared with the Sephadex G-25 columns showed the highest recovery with all three marker peptides. The peptide FFVAPFPEVFGK gave the most variable results of the three peptides used. The polypropylene resin columns provided the most consistent recovery regardless of the peptide used for measurement. All of the columns tested met the recovery requirements of SMPR 2016.002.

Conclusions
Many LC-MS/MS methods use isotope-labeled peptides as internal standards to allow quantification in multiple matrices, but we chose to use a matrix-matched external calibration curve because it provided acceptable accuracy in our samples as it accounted for losses in signal due to sample preparation and ion suppression. It was also more cost effective than using isotope-labeled internal standards, which must be custom made. Although internal standard addition and matrix matching can be used together (11,30), we found that matrix matching  alone provided good results. The method is intended to be used in a manufacturing setting in addition to food analysis laboratories; using matrix matching simplifies the implementation and use of the method on a day-to-day basis. We also found that overnight trypsin digestion without prior reduction and alkylation of samples provided good recovery, possibly because the marker peptides lack cysteine residues (31).The LC-MS method for casein allergens presented here can accurately and reliably quantify intact casein in infant formula made with extensively hydrolyzed casein. All three marker peptides for casein provided acceptable accuracy and precision under specific experimental conditions, even when different desalting columns were used. One limitation of the method is its inability to detect whey protein allergens, but by measuring casein allergens the method can detect intact casein from milk as well as any peptide fragments above 7 kDa that might be in the extensively hydrolyzed casein ingredient. Although it is possible that smaller peptides can cause an allergic reaction, most antigenicity in hypoallergenic formulas is associated with residual peptide fragments !10 kDa (32), making this method a good confirmatory test for extensively hydrolyzed casein infant formula.

Funding
There was no outside funding for this work.