Quantification of aflatoxin and ochratoxin contamination in animal milk using UHPLC-MS/SRM method: a small-scale study.

Mycotoxin contamination in animal milk is an emerging concern around the globe. Here we developed and validated an ultrahigh-performance liquid chromatography and mass spectrometry-selected reaction monitoring (UHPLC/MS-SRM) method to quantify low concentrations of aflatoxins (AFs) and ochratoxins (OTs) in routinely consumed animal milk samples collected from southern India. Stable isotope dilution methodology was applied to quantify AFB1, AFB2, AFG1, AFG2, AFM1, AFM2 and OTA, OTB in n = 38 different milk samples, using 1 mL of milk. Bioanalytical parameters including method accuracy, precision, recovery, regression analysis and stability were assessed. Dynamic ranges for quantification were between 15.6-1000 pg/mL for AFB1, AFB2, AFG1, and OTA; 7.8-500 pg/mL for AFM1, AFM2 and OTB; 78.6-5000 pg/mL for AFG2. Method accuracy ranged between 80-120%, with ± 15% precision. Recoveries for spiked standards were > 88% in water and 75% in milk, with limits of quantification (LOQ) ranging between 31.3 pg/mL for AFB1, AFB2, AFG1 and OTA, 15.6 pg/mL for AFM1, AFM2 and OTB and 156 pg/mL for AFG2. R2 values for regression analyses ranged between 0.9991-0.9999. AFB2 [mean: 38 pg/mL (0.038 µg/kg)] was quantified in goat milk, AFM1 was quantified in cow, goat, pasteurized milk [mean: 331 pg/mL (0.331 µg/kg), 406 pg/mL (0.406 µg/kg), 164 pg/mL (0.164 µg/kg)]. Additionally, 90% of cow, goat and pasteurized milk samples were above European Union (EU) limits of 50 pg/mL (0.05 µg/kg) and 40% of cow and goat milk samples were above the Food Safety Standards Authority of India (FSSAI) limit of 500 pg/mL (0.5 µg/kg). AFM2 was also quantified in cow, goat, and pasteurized milk samples [mean: 249 pg/mL (0.249 µg/kg), 375 pg/mL (0.375 µg/kg), 81 pg/mL (0.081 µg/kg)]. Our dynamic ranges for quantification are lower than other published methods, with need for a smaller volume of milk. This validated method can be applied for routine quantification of mycotoxins in milk. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s13197-021-04986-w).

Simple click chemistry-based derivatization to quantify endogenous formaldehyde in milk using ultra-high-performance liquid chromatography/tandem mass spectrometry in selected reaction monitoring mode.

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.

Maternal nutritional status and milk volume and composition in India: an observational study.

BACKGROUND: Human milk provides essential nutrition for infants, and its benefits are well established. We lack data on the influence of maternal nutritional status on milk volume and composition in low-middle income countries. OBJECTIVE: We aimed to 1) assess lactation performance (human milk volume, macronutrient composition, and infant energy intake) in Indian females and 2) examine the associations between maternal anthropometry (BMI, percentage body fat) and lactation performance. METHODS: We conducted an observational study among 232 mother-infant dyads, 2 to 4 mo postpartum in Haryana, India. We used deuterium oxide dose-to-mother technique to measure milk volume and maternal percentage body fat and collected human milk samples to determine macronutrient and energy concentrations. Adjusted multiple linear regression models were used to examine the associations between maternal anthropometry and lactation performance. RESULTS: The mean BMI and percentage body fat of mothers were 21.7 ± 3.6 kg/m2 and 29.5 ± 7.7, respectively. Milk volume and macronutrient composition were similar to the reference values (means ± standard deviations: milk volume, 724 ± 184 mL/d; median (25th, 75th percentile); protein, 9.9 (8.3, 11.7) g/L; fat, 41.0 ± 15.2 g/L; energy density, 0.71 ± 0.14 kcal/g; lactose, 65.5 (55.3, 71.3) g/L). Maternal BMI and percentage body fat were not significantly associated with macronutrient composition. Both maternal BMI and percentage body fat were negatively associated with milk volume (-7.0, 95% CI: -12.4, -1.6 mL/d; -3.5, 95% CI: -6.0, -1.1mL/d, respectively) but there were no effects on the total energy intake of infants after adjusting for covariates. CONCLUSION: Most mothers had a normal BMI and milk of similar composition and volume to reference values. Future work in populations with a greater burden of underweight and/or obesity are needed to examine the underlying mechanisms between maternal body composition and milk volume. This trial was registered at The Clinical Trials Registry- India as CTRI/2017/01/007636.

Quantification of B-vitamins from different fresh milk samples using ultra-high performance liquid chromatography mass spectrometry/selected reaction monitoring methods.

Absolute quantification of B-vitamins in milk is becoming imperative to correlate its impact on child/human health. In today's world of changing food habits and environmental pollution, there is concern if milk is what we think it to be. In the present work, ultra-high performance liquid chromatography mass spectrometry/selected reaction monitoring (UPHPLC-MS/SRM) methods have been developed and validated for quantitative estimation of 21 different B-vitamins [B1-3, B2-3, B3-2, B5-1, B6-5, B8-1, B9-2 vitamins, total choline (betaine, choline and acetylcholine)] from a drop (50 µL) and B12 from 500 µL of milk. These two methods qualify all essential bio-analytical parameters (recovery >80%, accuracy <±15%, variation <±10%) and are highly reproducible. By using these developed methods, B-vitamins in different fresh milk samples from cow, goat, buffalo and pasteurized cow milk (each 10 and total n = 40) were analysed. Total choline is the highest (6.5-10.5 µg/mL) and vitamin B8 is the lowest (16.1-32.9 ng/mL) in all milk samples. Inverse correlation between vitamin B2 and B5 was observed in milk samples. The ratio of vitamin B5/B2 was checked in all milk samples, it is higher in cow (2.64), equal in goat (1.04) and lower in buffalo (0.42) milk. Total B-vitamin content in cow milk is higher (10.5 µg/mL) compared to other three (goat-7.2, buffalo-6.5, pasteurized-8.8 µg/mL). Vitamin B12 is higher in cow milk (3.6 ng/mL) compared to other two fresh milk samples. Different isomers for vitamin B6 were noticed in the fresh milk samples. The complete profile of water soluble vitamins and the ratio of two abundant B vitamins (B5/B2) in milk will be useful to check the nutritional quality and to differentiate the kind of animal milk.