Rationale
Formaldehyde (FA) exposure via environmental pollution or through the food chain poses a serious threat to human health, especially in developing countries like India. Although the addition of FA to food is proscribed, it is often illegally added to foods such as milk to increase the shelf‐life. There are challenges in differentiating the endogenous FA content in milk from externally added FA.
Method
We have developed a simple method using ultra‐high‐performance liquid chromatography/tandem mass spectrometry in selected reaction monitoring mode (UHPLC/MS/SRM) for the absolute quantification of endogenous FA in milk. The steps include fat removal, protein precipitation using acid, and spiking with labelled FA (FA*), followed by simple click chemistry‐based derivatization using Girard P reagent (GP) and final analysis.
Results
A standard curve with FA* was constructed and used for the calculation of endogenous FA in milk. The optimal conditions for the derivatization reaction using 500 μL of milk were: GP, 50 μg; temperature, 37°C; time, 60 min; and 0.1% HCl. The validation parameters such as accuracy (95.84 to 99.73%), precision (2.84 to 8.02%) and spiked recovery (>95%) are within the FDA guidelines. This method is highly sensitive [limit of detection (LOD) of 1 ng/mL] with a dynamic range of 3.12 to 200 ng/mL. The endogenous FA level in pasteurized cow milk is 70 ng/mL (n = 60). The FA content in raw milk samples from cow, goat and buffalo (each n = 10) varied from 134 to 255 ng/mL.
Conclusions
This method is precise and sufficiently sensitive to quantify endogenous FA in milk samples using a minimal sample volume. As it involves simple sample preparation steps, it can be used routinely to quantify endogenous FA.
Objectives
Animal milk can be contaminated with mycotoxins (secondary metabolites of fungi) through poor quality feed and may be a source of human exposure. Our objective was to develop and optimize a method to detect biologically relevant concentrations of 8 mycotoxins (Aflatoxins B1, B2, G1, G2, M1, M2; Ochratoxins A, B) in animal milk.
Methods
We used ultra-high performance liquid chromatography/tandem mass spectrometry using selected reaction monitoring (UHPLC/MS-SRM) to quantify mycotoxins in animal milk samples (total N = 38; n = 10 each from cow and commercial milk and n = 9 from buffalo and goat) from the southern Indian states of Karnataka and Tamil Nadu.
Method development was conducted and stable isotope dilution employed, using AFB1-D3 for aflatoxins and OTA-D5 for ochratoxins. We validated the method and examined matrix effects, freeze-thaw and auto-sampler stability. Our dynamic ranges from quantification were between 7.8–5000 pg/mL.
Results
Among samples collected from Southern India, 8 of 10 cow [median 103.35 pg/mL; n = 3 > 500 pg/mL]; 0 of 9 buffalo and 10 of 10 commercial [median: 151.5 pg/mL], milk samples were above the LOQ. AFM2 was also seen in samples from both regions, but in lower quantities when compared to AFM1 [median (north): 25.8 pg/mL; median (south): 70.95 pg/mL].
All except 3 samples were below the LOQ (31.3 pg/mL) for OTA, however we detected a sodium adduct of OTA above LOQ, across samples. We found [Na-OTA] in goat milk [median: 5.9 ng/mL] > buffalo [median: 2.2 ng/mL] > commercial [median: 2.04 ng/mL] > cow [median: 0.8 ng/mL]. Other mycotoxins were seen in concentrations close to or below LOQ. We did not identify significant stability issues.
Conclusions
We developed a highly sensitive method with biologically relevant dynamic ranges for detection of mycotoxins in milk samples. We found AFM1, AFM2, and Na-OTA in milk samples from Southern India. Further studies with larger sample sizes are warranted to establish the extent of mycotoxin contamination in milk.
Funding Sources
Funded by University Research Committee, Emory University and International Society for Research in Human Milk and Lactation.
Background: Estimation of macronutrients like protein and lactose is important to assess the quality of milk. To estimate these two macronutrients, ten raw milk samples obtained from each group of different animals (cow, goat, buffalo), ten pasteurized cow milk and ten human milk samples were analysed. Methods: Bicinchoninic acid (BCA) method was used to estimate protein from different milk samples. Four different sample preparation protocols were compared to check the effect of fat on BCA based protein estimation: dilution (D), fat removal-protein precipitation (FR and PP), fat removal-dilution (FR and D) and dilution-fat removal (D and FR). For lactose quantification, ultrahigh-performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was developed and validated using 13C6 lactose as internal standard (ISTD).Result: Among these four different protocols, D and FR method showed consistent data for total protein content in animal milk (cow-3.16%, goat-3.21%, buffalo-3.81%, pasteurized-2.98%) and FR and PP showed consistent data in human milk samples (1.2%). Though BCA method is simple to use, proper sample preparation protocol has to be applied prior to protein estimation to avoid the interference due to fat or lactose. In case of lactose, inter-day validation showed the accuracy ranging from 97.13 to 100.54%, coefficient of variation varying between 0.1 to 1.53%, correlation R2=0.999. Lactose is in the range of 4.1 to 4.8% in animal milk and 6.6% in human milk samples. The internal ratio of lactose/protein (1.28 to 1.55 in animal milk and 5.33 in human milk) will be useful to differentiate human milk from animal milk type and to assess the milk quality.
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