What’s Your Pet Truly Eating? Analyzing Vitamins in Pet Food

If dogs are truly man’s best friend, shouldn’t we be more concerned with what we are feeding them?

The animal lovers at Phenomenex decided to dig a little deeper into our pet’s food, especially what kind of nutrition they are absorbing. The goal was analyze to Vitamin D2/D3 and Pre-D2/D3 pet food using LC-MS-MS. The same challenges persist in determining these vitamins in human foods and dietary supplements, such as matrix interferences, isomer conversions, and light sensitivity, as it does in pet food matrices.

Determination of vitamin d in pet food.

Vitamin D is an essential fat soluble vitamin, most known for its requirement in bone formation in animals amongst other multiple beneficial functions. Vitamin D3 is typically produced naturally with exposure to sunlight; however, it can be supplemented in diet if not adequately exposed.

In animals, Vitamin D3 is the most biologically active form, and needs to be distinguished apart from Vitamin D2, which is sourced from some plants and mushrooms. The isometric forms, pre-vitamin D2 and pre-vitamin D3, are also important to measure accurately without disrupting their natural balance to accurately assess their source.

Quantifying Vitamin D in complex pet food matrices is filled with a range of challenges, from isomer conversion to light sensitivity, saponification, and extraction efficiency.


Samples were partially protected from light with amber vials and dim lighting, with completion taking only 1 hour. The room temperature saponification was performed to minimize conversion to the pre-D forms for a more accurate quantification. After a double liquid-liquid extraction, a normal phase SPE protocol using Strata® NH2 cartridge was used to further remove some matrix components.

Near baseline resolution of Vitamin D and pre-vitamin D was achieved with a fast run-time on a short Kinetex® 2.6 µm F5, leveraging the polar and aromatic selectivity of the pentafluorophenyl ligand. The LC gradient was extended an additional two minutes with 100% organic solvent to elute some matrix components that might otherwise interfere in subsequent injections.

Quantification was done using a standard addition curve, inducing from a back calculated blank sample. Accuracy and precision were evaluated at two QC levels; accuracy of 99.2-106% with CV % of 3.23-8.61% for parent compounds of vitamin D2/D3, and accuracy of 89.8-110% with CV % of 2.32-9.01% for pre-vitamin D2/D3.

The application showed a quantitative method for analyzing Vitamins D2 and pre-vitamins D2 and D3 in dog food gave more opportunities to explore a wide range of food and pet food matrices.

We want our pets to stick around as long as possible, so making sure that they receive the right amount of vitamins from their food is crucial.

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