Glycosylated 1,25-dihydroxycholecalciferol from Solanum glaucophylum extract

In the current application an authorisation is sought under Article 4(1) for solanum glaucophyllum leaf extract (SGE) under the category/functional group (3a) "nutritional additives"/"vitamins, pro-vitamins and chemically well-defined substances having similar effect", according to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically, the authorisation is sought for the use of the feed additive for dairy cows for milk production and other dairy ruminants. 

According to the Applicant, the feed additive (SGE) is a powder preparation of ethanol extract of dried solanum glaucophyllum leaves. The active substance of the feed additive is glycosylated 1,25-dihydroxycholecalciferol with its content ranging from 50 to 160 mg/kg feed additive. 

The feed additive is to be used as a complementary feed in the form of controlled release bolus given to dairy cows or other dairy ruminants prior calving. The intended dose for dairy cows is 1 or 2 bolus corresponding to 500 μg of 1,25-dihydroxycholecalciferol per animal, while the dose for other dairy ruminants is 1 or 2 bolus corresponding to 1.25 μg of 1,25-dihydroxycholecalciferol/kg body weight of the animal. The feed additive is not intended to be used in premixtures or complete feedingstuffs. 

For the quantification of glycosylated 1,25-dihydroxycholecalciferol in the feed additive and complementary feed (bolus) the Applicant submitted a single-laboratory validated and further verified method based on analysis by high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) after an enzymatic hydrolysis with beta-glucosidase enzyme followed by derivatisation with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). 

The following performance characteristics were obtained for the quantification of the glycosylated 1,25-dihydroxycholecalciferol content in the feed additive and complementary feed (bolus) in the frame of the validation and verification studies: 

- for the feed additive with glycosylated 1,25-dihydroxycholecalciferol content ranging from 115.4 to 140.0 mg/kg: a relative standard deviation for repeatability (RSDr) ranging from 5.2 to 6.3 %; a relative standard deviation for intermediate precision (RSD_ip) ranging from 8.0 to 8.2 %; and a recovery rate (R_Rec) ranging from 95 to 103 %. 

- for bolus with glycosylated 1,25-dihydroxycholecalciferol content ranging from 28.3 to 99.3 mg/kg: RSD_r ranging from 3.3 to 4.8 %; RSD_ip ranging from 5.8 to 18.4 %; R_Rec ranging from 94 to 101 %; and a limit of quantification (LOQ) of 12.4 mg glycosylated 1,25-dihydroxycholecalciferol/kg of bolus. 

Based on the acceptable performance characteristics available, the EURL recommends for official control the single-laboratory validated and further verified method based on LC-MS/MS for the quantification of the glycosylated 1,25-dihydroxycholecalciferol content in the feed additive and complementary feed (bolus). 

Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005, as last amended by Regulation (EU) 2015/1761) is not considered necessary.

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2024/1685

For the quantification of glycosylated 1,25-dihydroxycholecalciferol in the feed additive and in the complementary feed (bolus):

• Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)